Western blotting at 40

On-demand webinar

Summary:

Join Dr. Hanna Dreja for the most recent know-how in western blotting. This technique may be middle-aged, but with being published on PubMed more than 400,000 times, it remains highly relevant. Learn how to get the most from your western blots. The webinar covers all you need from standard protocols and selecting the best reagents, to multiplexing and answers to your FAQs.

Webinar Objectives:

Describe the western blot process

Prepare suitable samples and controls

Explain how to identify which antibody to use

Western Blotting at 40-FINAL

Video Transcript

00:00 - 00:12: Thank you for joining Abcam’s webinar, Western Blotting at 40, an update.
00:12 - 00:18: During this webinar, we will cover the following objectives, outline the Western Blot process,
00:18 - 00:23: show you how to find suitable samples and controls, and finally teach you how to identify
00:23 - 00:25: which antibody to use.
00:25 - 00:29: Thank you and I hope you enjoy.
00:29 - 00:34: Welcome to Abcam’s webinar, Western Blotting at 40, an update.
00:34 - 00:36: And thank you for joining me today.
00:36 - 00:41: My name is Hanna Dreya and I lead the scientific quality control at Abcam.
00:41 - 00:46: I will base a lot of the information in this presentation on the work that my team has
00:46 - 00:52: been performing in the last few years, which has involved something like 5,000 Western
00:52 - 00:53: Blots.
00:53 - 00:58: But I will also draw on my own experience that I gathered during my many years of bench
00:58 - 01:02: work in an academic setting.
01:02 - 01:06: So I will start with a general introduction to Western Blot.
01:06 - 01:11: Then I will share a video of the practical Western Blot process, step by step.
01:11 - 01:17: I will go through suggestions on how to generate that ultimate Western Blot data, including
01:18 - 01:24: how to choose a good antibody, what type of samples to use, how this should be prepared,
01:24 - 01:29: what additional controls should be added, and what detection methods to choose.
01:29 - 01:33: I will also answer some questions that I frequently get.
01:33 - 01:41: And importantly, I will address your questions in an open Q&A session at the end.
01:41 - 01:49: So last year, this technique celebrated its 40th year, a technique that originated in
01:49 - 01:56: 1979 in the laboratory of a Harry Taubin in Basel, Switzerland.
01:56 - 02:03: There was a publication earlier this year by a Dr. Moritz, who stated that during these
02:03 - 02:09: 40 years, Western Blot has been mentioned in titles, abstracts, and keywords on more
02:09 - 02:13: than 400,000 med-listed publications.
02:13 - 02:18: It may actually still be the most used protein analytic technique.
02:18 - 02:24: So albeit middle-aged, it is still utterly relevant.
02:24 - 02:31: Amongst the protein detection techniques, Western Blot is unique as it can estimate
02:31 - 02:37: not only the presence, relative amounts, and modifications, but also the size of the target
02:37 - 02:38: protein.
02:38 - 02:45: So Western Blot relies on specific antibodies to identify proteins that have been separated
02:45 - 02:56: based on size by gel electrophoresis, where smaller proteins migrate faster than the larger ones.
02:56 - 03:06: After this, proteins are migrated to a membrane made of nitrocellulose or PVDF,
03:06 - 03:11: using electrophoresis.
03:11 - 03:17: The membranes can then be processed with antibodies specific for the target of interest and visualized
03:17 - 03:23: using labeled secondary antibodies and, if needed, detection reagents.
03:23 - 03:27: The signal can then be detected.
03:27 - 03:32: So rather than me trying to mimic the process from this screen, I will share a video with
03:32 - 03:38: you that captures the different steps in the process.
03:38 - 03:43: The purpose of Western Blotting is to separate proteins on a gel according to their molecular
03:43 - 03:44: weight.
03:44 - 03:49: The proteins are then transferred onto a membrane where they can be detected using antibodies.
03:49 - 03:54: Heat the samples at 95 degrees C for 5 to 10 minutes in a sample buffer containing a
03:54 - 03:58: reducing agent, such as beta-mercaptoethanol.
03:58 - 04:07: This results in linearized proteins with a negative charge proportional to their size.
04:07 - 04:11: Place a gel into the electrophoresis tank and add a buffer, ensuring the tops of the
04:11 - 04:13: wells are covered.
04:13 - 04:17: The acrylamide percentage of the gel being used depends on the molecular weight of the
04:17 - 04:21: target protein.
04:21 - 04:33: Load a molecular weight marker into the first lane.
04:33 - 04:37: Then load the samples into adjacent wells.
04:37 - 04:42: All the samples should contain equal amounts of protein.
04:42 - 04:52: Once all the samples are loaded, add a running buffer.
04:52 - 04:58: Place the lid onto the electrophoresis tank.
04:58 - 05:02: Turn on the power supply and set the voltage recommended by the manufacturer of the gels
05:02 - 05:04: in the gel tank.
05:04 - 05:08: You should be able to see bubbles rising through the tank.
05:08 - 05:17: Turn off the gel until the dye front has moved sufficiently down the gel.
05:17 - 05:22: The next stage is to transfer the proteins from the gel onto a membrane.
05:22 - 05:28: Membranes are usually made from nitrocellulose or PVDF.
05:28 - 05:37: Remove the gel from the tank and carefully release it from its plastic case.
05:37 - 05:54: Cut off the wells and the gel foot and place the gel into the transfer buffer.
05:54 - 05:58: Prepare the transfer stack by sandwiching the membrane and gel between filter paper
05:58 - 06:12: and sponges.
06:12 - 06:38: The membrane should be closest to the positive electrode and the gel closest to the negative
06:38 - 06:48: electrode.
06:48 - 07:10: Use a small roller to remove any bubbles between the gel and the membrane.
07:10 - 07:14: Clamp the transfer case closed and submerge it into a transfer tank containing transfer
07:14 - 07:31: buffer.
07:31 - 07:47: Add water to the outer chamber to keep the system cool and put on the lid.
07:47 - 07:51: Turn on the power supply to begin protein transfer.
07:51 - 07:57: Time and voltage require optimization, so check the manufacturer’s instructions for guidance.
07:57 - 08:02: Now that the proteins have migrated from the gel onto the nitrocellulose membrane, the
08:02 - 08:07: protein of interest can be detected with an antibody.
08:07 - 08:11: The membrane can be removed from the cassette and the molecular weight marker should now
08:11 - 08:12: be visible.
08:12 - 08:16: If required, the transfer of proteins can be confirmed by staining the membrane with
08:16 - 08:24: Ponceau S solution.
08:24 - 08:29: To prevent non-specific binding of the antibody, the membrane needs to be blocked.
08:29 - 08:35: Pour blocking buffer onto the membrane and agitate gently on a rocker.
08:35 - 08:41: Typically, this is done using a solution of 5% milk or bovine serum albumin, BSA, for
08:41 - 08:44: two hours at room temperature or overnight at 4 degrees C.
08:44 - 08:50: The time and type of blocking buffer should be optimized, so check the data sheet of the
08:50 - 08:54: primary antibody you intend to use for details.
08:54 - 08:58: After the membrane is blocked, remove the blocking buffer and add the diluted primary
08:58 - 09:11: antibody in the same solution.
09:11 - 09:14: Incubate on a rocker as before.
09:14 - 09:18: Typically primary antibody incubations are for one hour at room temperature or overnight
09:18 - 09:20: at 4 degrees C.
09:20 - 09:24: Antibody concentration and incubation time will need to be optimized.
09:24 - 09:28: Refer to the antibody data sheet for guidance.
09:28 - 09:34: Pour off the primary antibody and rinse the membrane twice in wash buffer.
09:34 - 09:42: Follow with one 15-minute wash and three 10-minute washes on a rocker.
09:42 - 09:48: The wash buffer is usually Tris-Buffered Saline, TBS, or Phosphate Buffered Saline,
09:48 - 09:53: PBS with 0.1% Tween-20.
09:53 - 09:57: Pour off the wash buffer and incubate the membrane in the conjugated secondary antibody
09:57 - 10:02: which has been diluted in blocking buffer.
10:02 - 10:06: Usually, this is done for one hour at room temperature, but antibody concentration and
10:06 - 10:17: incubation time will need to be optimized.
10:17 - 10:32: Pour off the secondary antibody and wash the membrane as shown previously.
10:32 - 10:35: There are several different systems for detection.
10:35 - 10:40: If the secondary antibody is conjugated to an enzyme, incubate the membrane in the appropriate
10:40 - 10:42: substrate before imaging.
10:42 - 10:46: If the secondary antibodies are fluorescent conjugates, then you can move directly onto
10:46 - 10:48: the imaging step.
10:48 - 10:55: Imaging can be carried out with x-ray film or with a digital imaging system.
10:55 - 11:04: Place the membrane into an imaging tray.
11:04 - 11:07: Place the imaging tray into the imaging system.
11:07 - 11:11: Exposure times will most likely need to be optimized in order to clearly detect the bands
11:11 - 11:26: relating to the proteins of interest.
11:26 - 11:33: So obviously, every immunoassay relies on a specific antibody, but how can you find
11:34 - 11:35: one that will work in your hands?
11:35 - 11:45: For example, on BioCompare’s website, I found 4,827 anti-TNF-alpha antibodies and on our
11:45 - 11:49: Abcam website alone, there are 45.
11:49 - 11:55: So before you start spending two days to decide which antibody to use, I recommend filtering
11:55 - 11:57: your searches.
11:57 - 12:02: So in the frequently asked questions section later, I will share some search tools that
12:02 - 12:04: you may find useful.
12:04 - 12:09: So firstly, has the antibody been demonstrated to work in Western blot?
12:09 - 12:16: If so, do check the supplier’s website and importantly, question the data.
12:16 - 12:21: If they have only used the recombinant protein to demonstrate signal in Western blot, be
12:21 - 12:27: cautious if you are interested in using endogenous samples with low levels of expression.
12:27 - 12:29: Does the size make sense?
12:29 - 12:32: Did they include positive and negative controls?
12:34 - 12:38: And I know negative controls are not necessarily easy to obtain.
12:39 - 12:45: And to really demonstrate the specificity for our antibodies, Abcam has, we are rigorously
12:45 - 12:47: testing them using knockout samples.
12:48 - 12:52: So where the samples have been engineered to not express the protein of interest.
12:53 - 13:00: This does help to demonstrate that the antibody is specific and indeed of the correct size.
13:00 - 13:06: And as you can see, we do get a complete lack of signal in our knockout sample name here.
13:08 - 13:16: So the next thing that I would recommend is ask if other scientists have been successful and
13:16 - 13:20: look at references and publications.
13:21 - 13:25: You can see a number of the references on the supplier’s website.
13:25 - 13:29: So for this particular example, you can see it’s stated up here.
13:31 - 13:36: CiteAb reports on the number of citations for each antibody.
13:38 - 13:43: And this can be a good way to direct your interest, but do be cautious.
13:43 - 13:49: A 30-year-old antibody would have accumulated significantly more citation, of course,
13:49 - 13:51: than a recent superior one.
13:52 - 13:57: And there are other websites as well, such as obviously PubMed and Labome, etc.
13:59 - 14:05: And then take the chance to look at other scientists’ reviews.
14:06 - 14:11: Again, going back to the supplier’s website is very useful.
14:11 - 14:17: In this particular case, this product has received 16 reviews.
14:18 - 14:20: And we have this here at Abcam.
14:20 - 14:24: And this is an uncensored reporting.
14:24 - 14:27: It’s basically like a trip advisor for our antibodies.
14:27 - 14:32: It doesn’t necessarily mean that all these 16 reviews are for Western blots.
14:32 - 14:34: So please click around and see.
14:34 - 14:40: And it’s really useful because these reviews may also provide some great suggestions too,
14:40 - 14:42: to get your antibody to work.
14:43 - 14:47: Lastly, actually, probably not lastly, but a very important thing.
14:47 - 14:52: Are you expecting to work with this antibody over a period of time?
14:54 - 14:56: This is possibly a bit controversial.
14:57 - 15:04: But all that previous work you have done researching your antibody may be pointless
15:04 - 15:10: if you choose a polyclonal antibody, as their performance can differ significantly between
15:10 - 15:11: batches.
15:11 - 15:15: Of course, as a supplier, we test every batch in the same manner.
15:16 - 15:22: But there’s always small differences between batches that may have been generated in different
15:22 - 15:23: animals.
15:23 - 15:29: So in one of my previous lab posts, we purchased one of the few antibodies that were available
15:29 - 15:32: for an obscure and unknown target.
15:33 - 15:34: It worked great.
15:34 - 15:36: Six months later, the new batch came in.
15:36 - 15:39: And we could not repeat that initial finding.
15:39 - 15:43: But we had to actually go and try to make our own antibody.
15:43 - 15:49: So monoclonals will provide you with a reassurance of limited batch-to-batch variability,
15:50 - 15:55: which of course is essential if you want to continue to do experiments to detect that
15:55 - 15:57: protein beyond your first file.
15:58 - 16:05: So I will go beyond that and would recommend a recombinant monoclonal antibody, which would
16:05 - 16:10: have very minimal difference between the lots due to them being genetically identical.
16:11 - 16:14: Next, sample choice.
16:14 - 16:15: Know your target.
16:16 - 16:22: I guess I could have, this could have been my first slide because if you do not know
16:22 - 16:26: exactly what you’re looking for, you will struggle to design a good experiment.
16:27 - 16:32: I will go into more detail again during the frequently asked questions session.
16:33 - 16:39: So do you expect to see this in normal cells, or do they require stimulation?
16:39 - 16:46: Are you interested in a yes or a no answer, or do you want to know if the protein is predominantly
16:46 - 16:48: in a nucleus or in the membrane?
16:49 - 16:52: Control, please include a positive control.
16:52 - 16:56: Something that guarantees expression of the protein of interest.
16:56 - 17:03: This will make your life a lot easier to have that particular signal there.
17:04 - 17:07: Negative control.
17:07 - 17:14: As I mentioned, it’s not always easy to come by if you are looking for a ubiquitously
17:14 - 17:15: expressed protein.
17:16 - 17:24: So to address this and provide material, Abcam has now over 2,500 lysates in our catalog,
17:24 - 17:26: where the gene has been knocked out.
17:26 - 17:30: This is a good resource helping you validate your experiment.
17:32 - 17:33: This is one of the examples.
17:33 - 17:39: Again, you see the lack of signal using that knockout lysate sample.
17:40 - 17:46: We have also started to sell cell lines, which of course can be essential if you want to
17:46 - 17:52: do experiments to understand what the phenotype is in the cells that do not express the protein
17:52 - 17:53: of interest.
17:54 - 17:58: And do you know which size you are looking for?
17:59 - 18:04: Using the expected molecular weight based on the amino acid sequence can often give
18:04 - 18:06: you a significantly wrong estimate.
18:07 - 18:12: So do check other reports, publications and understand if there are post-translational
18:12 - 18:16: modifications that could affect the molecular weight.
18:18 - 18:25: Once you know which sample to include and have a short list of antibodies, then it is
18:25 - 18:28: time to decide on the sample preparation.
18:29 - 18:37: So just a brief reminder, denaturation, this disrupts interactions responsible for secondary
18:37 - 18:37: structures.
18:37 - 18:41: And we do this by LDS or SDS.
18:42 - 18:49: Reducing conditions, this breaks disulfide bridges in proteins so that they can adopt
18:49 - 18:55: random conformation and be better separated by the SDS-PAGE gels.
18:56 - 19:00: This means using beta-mercaptoethanol or DTT.
19:01 - 19:06: The majority of the time you will be using denatured and reduced samples, but there are
19:06 - 19:13: antibodies that require native conditions and this should be stated in the supplier’s
19:13 - 19:14: information pack.
19:15 - 19:17: Control your experiments.
19:17 - 19:21: I have spoken about choosing the control samples.
19:21 - 19:26: What else do I suggest to get that ultimate publication-worthy result?
19:27 - 19:34: As you likely want to compare samples, demonstrating that the same amount of protein is loaded
19:34 - 19:35: is important.
19:35 - 19:39: So you can do this by a number of different ways.
19:39 - 19:42: Staining the membrane for proteins will allow for this.
19:42 - 19:49: And using fluorescent secondary antibodies, so you can use two antibodies on the same
19:49 - 19:50: blot.
19:50 - 19:54: This is really useful if you want to see the bands in different colors.
19:55 - 20:00: And also you can use this approach if you’re interested in modified versions of your proteins
20:00 - 20:01: too.
20:02 - 20:09: You can cut the membrane if you know that the band of interest is significantly different
20:09 - 20:10: than the molecular weight of that loading control of yours.
20:10 - 20:19: You can strip the blot and reprobe it with an anti-loading control antibody and you could
20:19 - 20:21: run a separate gel for the loading control.
20:22 - 20:30: Of course, if the gels did not run or transfer identically, this will never be a true control.
20:31 - 20:33: Next, sample quantity.
20:33 - 20:39: If you’re looking for more than a yes or no answer, but rather want a semi-quantitative
20:39 - 20:44: outcome, you should titrate your sample input so that your signal is indeed reflective of
20:44 - 20:45: the amount loaded.
20:46 - 20:50: And measuring an oversaturated band will not help you.
20:51 - 20:53: Blocking and dilution buffers.
20:54 - 20:56: The strength of the blocking buffer is important.
21:02 - 21:09: If your results are too vague, consider using non-fat milk if the signal is too weak.
21:09 - 21:11: BSA could be the answer.
21:11 - 21:15: There are plenty of non-mammalian based blockers that work really well too.
21:15 - 21:18: So do check what works best in your hands.
21:19 - 21:21: Antibody dilution.
21:22 - 21:27: The supplier generally provides you with a range, but be prepared that you may have
21:27 - 21:30: to check a few different dilutions to get that ultimate signal.
21:31 - 21:32: And incubation time.
21:33 - 21:37: Longer generally gives you more signal, to a certain degree, of course.
21:38 - 21:46: If overnight is your choice, do this at refrigerated temperature.
21:47 - 21:48: Detection system.
21:48 - 21:52: There are a few things to consider when choosing the detection system.
21:53 - 21:57: And the most relevant question is likely, what do you have in your lab?
21:58 - 22:04: So in chemiluminescent detections, you use an antibody attached to an enzyme,
22:04 - 22:06: horseradish peroxidase or alkaline phosphatase.
22:08 - 22:14: This triggers a reaction with a luminescent substrate, which in turn produces light as
22:14 - 22:15: a byproduct.
22:16 - 22:24: This is detected by exposing the membrane to an x-ray film, or you can scan the membrane
22:24 - 22:28: on a charge-coupled device imaging system, which captures that signal directly.
22:29 - 22:36: The advantages is, while using film is considered the most sensitive system and is therefore
22:36 - 22:39: good for low-expressed protein.
22:41 - 22:47: Disadvantages, but multiplexing is difficult as there’s no way to distinguish bands of
22:47 - 22:48: similar size.
22:49 - 22:54: As I mentioned previously, the membrane can be stripped and reprobed, but this can reduce
22:54 - 22:54: your signal.
22:55 - 23:01: My top tip would be to work quickly as you’re using an enzymatic reaction, as you need
23:01 - 23:07: to capture that signal as it is happening on the membrane.
23:08 - 23:13: With film detection, try a few different exposure times to obtain that optimal image for
23:13 - 23:14: quantification.
23:15 - 23:20: And as I mentioned, the CCD imaging is simpler and can also be automated.
23:21 - 23:27: If you’re struggling to get a band, try perhaps a different ECL substrate.
23:29 - 23:39: The fluorescent detection method, here are specific fluorophores conjugated to the secondary
23:39 - 23:44: antibody, so no additional substrate is necessary.
23:44 - 23:49: The fluorophores are excited by infrared light and the signals captured and quantified by
23:49 - 23:51: a digital imaging system.
23:52 - 23:59: The advantage is, while it’s stable, it has a much greater dynamic range and you can perform
23:59 - 24:00: multiplexing.
24:01 - 24:06: This multiplexing has been essential for our knockout validation as we can demonstrate
24:06 - 24:12: that we have loaded similar quantities of proteins in the control versus the knockout
24:12 - 24:14: lane, as you can see up here.
24:16 - 24:23: My top tips, choose secondary antibodies with different fluorophores for that multiplexing.
24:25 - 24:27: As I mentioned, there’s stability.
24:27 - 24:33: Store your membrane away from the light and you could re-image them a few years later,
24:33 - 24:34: even if you ever would need to.
24:37 - 24:45: This summary slide is taken from a very good review published earlier this year by Sam
24:45 - 24:45: Heaton.
24:49 - 24:55: This is really helpful, summarizing how to choose the correct antibodies, how to demonstrate
24:55 - 25:02: specificity by using correct controls, how to verify selectivity.
25:02 - 25:08: This means the preference of an antibody to bind to one target over others and one
25:08 - 25:11: can do that by optimizing the experiments.
25:13 - 25:19: It also talks about reproducibility, but one of the things that is stressed is the importance
25:19 - 25:21: when reporting and publishing data.
25:21 - 25:27: So please, all your researchers, do you provide all essential information for others to be
25:27 - 25:28: able to reproduce your work?
25:29 - 25:33: So Western blotting remains in the spotlight of publications.
25:34 - 25:38: There have been recent developments in Western blotting to make this even more relevant,
25:39 - 25:48: such as making the application faster, miniaturization experiments and incorporating automation.
25:49 - 25:56: For example, Western blot can be done by capillary electrophoresis and microfluidic Western
25:56 - 25:59: blot may also be another option.
26:00 - 26:04: I’m sure we will see many more developments in the near future to come.
26:07 - 26:09: So frequently asked questions.
26:10 - 26:15: I will now capture questions that I have gathered in advance of this talk.
26:16 - 26:22: First of all, where can I find antibodies that I can use apart from on the supplier’s
26:22 - 26:23: website? is the question.
26:24 - 26:33: And again, as I mentioned, this publication is useful and we have a few different options
26:33 - 26:37: and search engines and directories that I can recommend that you would look at.
26:39 - 26:43: How can I identify positive and negative controls for my experiments?
26:43 - 26:51: And again, there is a really useful table to have a look at, to look at protein RNA
26:51 - 26:54: expression profiles in this review.
26:55 - 27:00: Why are the Western blot band sizes different from what is expected?
27:01 - 27:08: Post-translational modification, particularly glycosylation, can increase the size of a
27:08 - 27:08: protein.
27:09 - 27:12: Then we have the post-translational cleavage.
27:13 - 27:19: Many proteins are synthesized as pro-proteins and then cleaved to give the active form.
27:19 - 27:27: For example, we have the pro-caspases, and so therefore you can expect to see different
27:27 - 27:31: smaller sizes of the same protein.
27:32 - 27:39: Splice variants and isoforms, this is due to alternate splicing and it can create different
27:39 - 27:41: size proteins produced from the same gene.
27:42 - 27:48: Relative charge, the composition of the amino acids can affect the way the protein runs on
27:48 - 27:49: the gel.
27:50 - 27:53: Multimers, so dimerization of the protein.
27:54 - 28:00: This is usually prevented in reducing conditions, although strong interactions can result in
28:00 - 28:02: the appearance of higher bands.
28:03 - 28:06: Next, how much samples should be loaded?
28:07 - 28:13: Well, I would recommend somewhere between 10 to 50 micrograms of total protein per well.
28:14 - 28:22: Of course, you would need to check and possibly titrate that to get it right and for it to
28:22 - 28:24: suit your testing conditions.
28:25 - 28:31: If you are using purified proteins, of course, you need to load significantly less.
28:31 - 28:37: I would probably try in the range of 10 to 100 nanograms of that protein.
28:39 - 28:42: Next, do the samples need to be reduced and denatured?
28:43 - 28:46: Well, I recommend reviewing the data sheet for further details.
28:48 - 28:53: For the majority of our antibodies, we test them under reducing and denaturing conditions.
28:53 - 29:00: But there are cases in which this is not suitable, and that would be stated on our website.
29:03 - 29:06: Blocking, is there a difference between MILK and BSA blocking?
29:06 - 29:14: Well, MILK is a stronger blocking solution, and it generally results in less background
29:14 - 29:14: signal.
29:15 - 29:20: However, some antibodies can be sensitive to the blocking reagent, and some antibodies
29:20 - 29:21: will work better in BSA.
29:22 - 29:29: So, if you’re not happy with the first set of results you have, I would recommend to
29:29 - 29:34: go back and try another blocking solution.
29:35 - 29:40: Can I incubate my primary antibody at room temperature for one hour instead of four degrees
29:40 - 29:41: overnight?
29:43 - 29:48: Well, again, this can be optimized and should be optimized by the end user.
29:48 - 29:52: It’s great if you have all that flexibility.
29:52 - 29:56: Sometimes, for practical reasons, you may need to go home at one point and have dinner,
29:56 - 30:01: so four degrees overnight is not necessarily a bad option.
30:02 - 30:13: If you want to join me for a live Q&A session, please join me on the 11th of June, and this
30:13 - 30:16: is at 3 o’clock UK time.
30:18 - 30:26: If you want to submit your questions beforehand, please email eventsteam at abcam.com.
30:28 - 30:34: Thank you all for listening in today, and I hope it has been useful.
30:34 - 30:40: Please feel free to contact us at any time, and hopefully we can help you with whatever
30:40 - 30:41: questions you have.
30:41 - 30:47: I also appreciate any suggestions and comments so that we can improve for our
30:47 - 30:48: next webinar.
30:48 - 30:50: Thank you all and have a great day.
30:50 - 30:56: Bye.

Western blotting at 40 live QA

Video Transcript

00:00 - 00:16: So hi everyone, so thank you for joining Abcam’s live Q&A conversation for our Western Blotting
00:16 - 00:23: at 40 webinar. Yeah, I hope you’ve all had a chance to watch the on-demand video describing
00:23 - 00:30: Western Blotting etc. So and yeah, during today’s session I’m joined by Dr. Hannah Dreyer
00:30 - 00:36: who’s here to answer all of your questions. So to get us started, we’ve received a number
00:36 - 00:40: of questions through our inbox, so I’m just seeing them all popping up here, which is
00:40 - 00:44: absolutely fantastic. So yeah, anytime you want you can answer a question through the
00:44 - 00:48: Q&A chat box, that’s absolutely fine. And if we’re unable to answer these, what we’re
00:48 - 00:52: going to do is going to collate them all and we’re going to answer them all individually
00:52 - 00:56: via email afterwards. So yeah, so at this time then I’ll hand it over to Hannah, who’ll
00:56 - 01:01: give you a brief introduction. Yes, hi, thank you very much Simon. Yes, so my name is Hannah
01:01 - 01:07: Dreyer and I work for Abcam. I run a team called Scientific Quality Control and we focus
01:07 - 01:12: on the quality of our antibodies, our products, ensuring that they are specific. And the way
01:12 - 01:21: my team assesses specificity is predominantly through Western Blotting. So I made a rough calculation
01:21 - 01:26: and I think in the last five years or so of knockout validation, we’ve done something
01:26 - 01:31: similar to 7,000 Western Blots. I haven’t done 7,000 Western Blots, but I’ve had lovely
01:31 - 01:36: colleagues helping me with it. So hopefully I’ll be able to share some of that knowledge
01:36 - 01:42: with you here today. And yeah, so yes, thank you, Simon, for the introduction and over to you.
01:42 - 01:46: Excellent, Hannah. Okay, shall we get started with some questions?
01:47 - 01:57: No problem. Okay, so there is a question from an anonymous attendee here. They haven’t put
01:57 - 02:03: the name, which is fine. So is there such a thing whereby the primary antibodies are
02:03 - 02:08: conjugated with fluorophore? If this exists, does it mean that secondary antibodies are
02:08 - 02:14: no longer useful? Well, I know Simon really wants to answer that question, but I’m going
02:14 - 02:19: to jump in there. So actually here, we do have something that we refer to as primary
02:19 - 02:25: conjugates. So this is exactly that. So this is where we have put a label on our primary
02:25 - 02:33: antibodies so that we don’t need those secondary antibodies. And obviously, this makes the
02:33 - 02:41: life so much easier for the researchers and it works really well. The important thing
02:41 - 02:46: here is obviously that our primary antibody needs to be very specific because you do not
02:46 - 02:53: get that amplification of signal as you would with your secondary antibody. But then our
02:53 - 03:02: antibodies, they rely on our RabMAb antibody portfolio. So they are indeed specific. So
03:02 - 03:10: we therefore have a range of different fluorophores that we can use for flow cytometry, immunocytochemistry,
03:10 - 03:18: immunohistochemistry. And in terms of Western Blotting, yes, they can also be used for Western
03:18 - 03:25: Blotting if you choose those wavelengths that are compatible with your reading device or
03:25 - 03:32: your scanner. We can also put on such a thing as not a fluorophore, such as horseradish
03:32 - 03:40: peroxidase, which is the enzyme that we would normally use for Western Blotting if you
03:40 - 03:47: are using the ECL detection system. And this works really, really well. So it just makes
03:47 - 03:52: your experiment really quick and fast, actually, because you only need to add one antibody.
03:52 - 03:59: So yes, we can do that. And I would warmly recommend to have a look at our antibody catalog
03:59 - 04:06: for those. Simon, do you want to add anything? Because I know that in your imaging experience,
04:06 - 04:11: you are using a lot of these primary conjugates. We are, yes. I was just going to mention the
04:11 - 04:16: fact that, yeah, with, of course, if you’re using a primary conjugated antibody, you haven’t got
04:16 - 04:21: the degree of signal amplification that you have with a secondary and a primary. So Hannah
04:21 - 04:26: already pointed out that, you know, your protein does need to be abundant enough, if you like,
04:26 - 04:32: for it to be detected using a primary conjugated antibody. And yes, always use fluorophores there
04:32 - 04:37: that your imaging system has the filter sets for. Otherwise you’re not going to see very much. So,
04:37 - 04:41: yeah. Thank you, Hannah. Thanks. Is there anything else you’d like to add, Hannah, or shall I?
04:42 - 04:53: You can continue. Okay. No problem. Okay. So Anastasia is asking, I do not believe that
04:53 - 05:00: housekeeping proteins give all time reliable results. Protein staining the blot and quantitating
05:00 - 05:08: the whole lane is more reliable. What do you think of this? I think, Anastasia, that you are
05:08 - 05:14: correct because it is great assuming that a housekeeping gene is expressed at exactly the
05:14 - 05:21: same level in each cell line. And that is not necessarily true. Absolutely. And also you’re,
05:22 - 05:28: you are relying on yet another antibody. So I do believe that measuring the protein
05:28 - 05:35: and stain is a very good option. Absolutely. It’s, it’s a very, it’s a nice alternative that
05:35 - 05:42: you can do for sure. So yes, if that is what you would prefer doing, I would recommend doing so.
05:43 - 05:50: We here at Abcam tend to use our loading controls mainly because we have a real confidence in that
05:50 - 05:57: our antibodies do work really well. And so it allows that visualization, which sometimes is
05:57 - 06:03: very useful if you want to multiplex and use that loading control together with your protein
06:03 - 06:11: of interest. But yes, you’re absolutely right. Just doing a protein stain is a very good
06:11 - 06:21: alternative. Okay. Thank you, Hannah. Thank you. And yeah, Anastasia is being quite prolific here.
06:21 - 06:27: So she also wants to know how many times is it okay to use diluted primary and secondary
06:27 - 06:32: antibody that we’ve prepared before? So can you reuse your primary and secondary antibodies in other
06:32 - 06:43: words? So this is interesting because obviously I have, I have the advantage working for Abcam
06:43 - 06:49: where I actually have a good supply of antibodies and I can, I don’t really need to reuse my
06:49 - 06:55: antibodies. We do have one or two, don’t we, Hannah? Yes, we do have them. And if I really
06:55 - 07:03: need one, I could always get one from the basement. But I have spent 12 years in academia
07:03 - 07:12: in different laboratories where I have had to, or I’ve chosen to reuse my antibodies. And we don’t,
07:13 - 07:19: we don’t recommend it, but I know it is happening. And if it works in your hands,
07:20 - 07:26: yeah, do it, but be prepared that it would be very difficult for you to compare the first
07:26 - 07:34: blot you do to the blot you do after having defrosted that milky solution four weeks later.
07:34 - 07:42: It’s not going to be exactly compatible, but if it’s a loxazy and it is, I can understand that
07:42 - 07:47: people would want to do that. We obviously recommend to do it fresh because that will allow
07:48 - 07:56: the test or retest to be much more compatible because you don’t have to take into consideration
07:56 - 08:02: the risk of the antibody going off, the milk going sour, et cetera. So I would not necessarily
08:02 - 08:06: recommend it, but I do understand when people do it, ensure you have your proper control and you
08:06 - 08:20: are prepared to accept that it’s not the ideal process to go down. How about in imaging? Do you
08:20 - 08:26: ever, is that something that you have been asked as well, Simon, if people can reuse diluted
08:26 - 08:34: antibodies? We never reuse diluted antibodies. We tend to, we use just enough for the actual
08:34 - 08:39: experiment. So we always make those dilutions up fresh. So what we do as well, some of the
08:41 - 08:47: higher dilution antibodies can actually, after sort of a few days, they can stop working.
08:47 - 08:51: We think there’s not enough protein stabilizer in there. You know, the overall protein content
08:51 - 08:55: isn’t high enough and maybe they become hydrophobic and fall out of solution or
08:55 - 09:00: something along those lines anyway. So it’s always good to dilute antibodies for any
09:00 - 09:05: immuno application, regardless of what it is. It’s always best if you can to make them fresh.
09:07 - 09:16: Thank you. Excellent. Thank you, Hannah. Okay. Question here from Mario. So when storing primary
09:16 - 09:20: unlabeled antibodies in a frozen form, does it make a difference if the liquid antibody
09:20 - 09:26: stock solution is frozen slowly at minus 20 degrees or quickly on dry ice?
09:27 - 09:34: This is something that I’m not actually going to be able to answer because I don’t know.
09:34 - 09:42: I’ve only just done it slowly at putting it at minus 20 and let the antibody freeze. So I’m not
09:42 - 09:48: sure exactly if it makes a difference. I can tell you that freezing it at minus 20 is certainly
09:48 - 09:53: not harmful for the antibody. That is the process we are using in the lab.
09:54 - 10:02: Okay. Thank you, Hannah. Okay. So Saruthi would like to know, could you please explain how to
10:02 - 10:09: determine the antibody dilution, especially for a secondary antibody? Okay. How to determine the
10:09 - 10:15: antibody dilution, meaning that how to dilute, this is how I interpret it, how to dilute
10:16 - 10:21: the secondary antibody or to determine which dilution one should use.
10:23 - 10:28: Sorry. It says determine the antibody dilution. So it’s working dilution, I think.
10:28 - 10:34: Okay. Okay. How to determine the working dilution. So obviously, as a provider of secondary
10:34 - 10:42: antibodies, we also provide a range of dilutions that we would recommend. And in the ideal scenario,
10:42 - 10:46: you should be able to stick to one dilution of the secondary antibody regardless of your target and
10:46 - 10:52: your primary antibody and everything would work fine, but which is obviously not the case. And
10:52 - 11:01: that’s why we are providing a range of dilutions. So I would warmly recommend having a look at that
11:01 - 11:08: range. And if you want to, if you have the opportunity, you can titrate it whilst also
11:08 - 11:13: titrating your primary antibody. But I can also understand that for most of you,
11:14 - 11:20: you want it to just grab and go. So if you just set your, if you do a range of different dilutions
11:20 - 11:26: and realize that actually it does work at one in 2,000, as well as one in 5,000, one in 20,000,
11:26 - 11:32: maybe you want to just sit somewhere in the middle so that you could repeatedly use exactly the same
11:32 - 11:37: secondary antibody at the same dilution to make your life easy. So in the ideal scenario,
11:37 - 11:43: titrate it and see where you can go. Otherwise, I would probably, as a go-to suggestion,
11:43 - 11:51: somewhere in the middle of that range is probably where I would go. So yes, and once you have
11:51 - 11:58: established that, you can then use that dilution range or that dilution. And
11:59 - 12:10: what I find is if you get, if your signal is too low, my first go-to would probably be adjust
12:10 - 12:15: the primary antibody concentration and keep that secondary antibody concentration stable.
12:15 - 12:21: And that again, it could be a case-to-case scenario where you would need to go up because
12:21 - 12:30: your primary antibody is not, it may not help increasing that beyond your first set of
12:30 - 12:37: concentrations. But generally, my go-to would be try something in the middle range and see if it
12:37 - 12:43: works. How do you do with your, do you titrate yours, of course, with imaging as well? Do you
12:43 - 12:48: concentrate on that, doing that, Simon? We always do titrations, Anna. That’s the
12:48 - 12:52: only way to get the optimal between the positive staining and the background.
12:54 - 12:55: And you do both of them, of course.
12:55 - 12:59: Spending that time and effort, and of course, money to do that. It really is.
13:03 - 13:09: Yeah. And just to let you know, so as I mentioned, we have done thousands of
13:10 - 13:14: Western blots for our knockouts. We have established where we are lying with our
13:14 - 13:21: secondary antibodies. So honestly, we haven’t needed to change our dilution of those secondary
13:21 - 13:26: antibodies for thousands of blots. What we tend to modify is the primary, as we know,
13:26 - 13:32: is such a good secondary antibodies that we are using. And we are currently, as you can see on
13:32 - 13:37: the legend on our website, we’re using them at one in 20,000. So I don’t move very much around
13:37 - 13:42: from that. But it’s very dependent on your secondary antibody and how reliable they are.
13:43 - 13:50: Excellent information. Thank you, Hannah. Okay. We have a question from Shajadl. Now,
13:50 - 13:53: I do apologize if I pronounce your name incorrectly there. I do apologize.
13:54 - 14:01: But why are antibodies sometimes fermented, brackets yellowish color, after incubating
14:01 - 14:07: overnight, particularly those antibodies diluted with skimmed milk powder? What protective measures
14:07 - 14:10: should I take to prevent it? Very interesting question.
14:11 - 14:19: That is an interesting question. Fermentation and milk. Yes. I have the pleasure of not having
14:19 - 14:27: seen that in my lab, actually. So I do wonder whether that is due to the quality of the milk.
14:27 - 14:36: I’m not sure because ours tend to stay white. That answer does not help you at all. I know that
14:36 - 14:43: for sure. But it may be worth looking into what kind of nonfat milk powder you are using.
14:50 - 14:55: I don’t know whether it could be anything to do with the pH. I’m not sure. But I’m hoping that
14:55 - 15:05: you’re using it with a buffered solution. So this would be TBS or PBS, which should balance the pH.
15:07 - 15:12: But yes, I also strongly recommend doing this, of course, at four degrees as well,
15:12 - 15:18: so nothing else is happening. So I don’t think I answered that particularly well,
15:18 - 15:25: because I actually don’t know. And I haven’t really seen that. But if it’s okay with you,
15:25 - 15:31: and I have now forgotten the name, but I have still the question here. Can I get back to you?
15:31 - 15:37: I will investigate why sometimes the milk can go off and turn yellow. Is that okay? Well,
15:38 - 15:40: I hope that’s okay. So I’ll get back to you on that one.
15:41 - 15:46: Thank you very much, Hannah. Yeah. Lots of questions coming in from Anastasia again,
15:46 - 15:53: which is fantastic. So let’s have a look. So Anastasia would like to know,
15:53 - 16:00: for Western Blotting phosphorylated proteins, can I use one times PBST for washing?
16:01 - 16:13: Yes. So the question is, can you use one times PBST for washing? Yes, that’s great. That’s what
16:13 - 16:22: we use all the time. You can modify your tween concentration if you want to. And obviously,
16:22 - 16:29: increasing the tween concentration would reduce the signal and possibly help if you have background
16:29 - 16:38: problems, you can modify that. But PBST or TBST, these are the go-to buffers that we would use for
16:38 - 16:46: our Western Blots. And also, of course, for the washing steps. What we tend to do here is utilize
16:46 - 16:53: the same washing buffer as we use for the antibody incubation, but with the addition of proteins,
16:53 - 17:00: so in most cases milk, that we would then re-suspend in the PBST or the TBST. But yeah,
17:00 - 17:06: those are suitable washing buffers. I think what Anastasia might be getting at there
17:07 - 17:12: is the phosphate in the buffer, because the antibody towards the phosphorylated protein,
17:12 - 17:19: is the phosphate in the buffer going to negatively affect the reaction in any way? Because I think
17:19 - 17:25: when you’re using an alkaline phosphatase label in IHC, for example, the phosphates in the buffer
17:25 - 17:30: can actually, I think, quench the alkaline phosphatase activity. If I remember correctly,
17:30 - 17:35: that’s off the top of my head, but I think it’s along those lines. Absolutely. It’s something to
17:35 - 17:41: take into consideration. In the past, I’ve never had any problems myself with that. However,
17:42 - 17:46: it may be that I would have been very fortunate with my proteins that I was looking at or the
17:46 - 17:53: antibodies being very strong. So if you have any doubts, going to Tris-buffered saline
17:54 - 18:01: may be the safer option. Here, when I run our blots, we are using TBST rather than
18:01 - 18:11: PBST, but I know a lot of our customers use PBST without any major issue. The issue, what you could
18:11 - 18:18: find if you’re working with phospho targets, is how do you choose that blocking solution?
18:19 - 18:25: Again, there’s a lot of debate around this. Can I use milk? Shouldn’t I use milk? Because milk
18:25 - 18:31: contains casein, and casein has a lot of phosphopeptides, basically. So there’s a lot of
18:33 - 18:40: noise in there that your phospho-specific antibody could potentially bind to, and that could produce
18:40 - 18:49: signal or cause background if the casein is obviously blocking your membrane. But I also
18:49 - 18:55: have customers that say that they don’t have any problems with that. But if you’re working with a
18:55 - 19:04: phosphoprotein, I would consider using another blocking agent than anything milk-related.
19:05 - 19:10: If you’re still concerned, TBST may be the safest bet, albeit I don’t have any
19:10 - 19:16: strict evidence that the PBST doesn’t work. Thank you very much.
19:17 - 19:24: Okay, so Dr. Anonymous would like to know, what is the function of a blocking buffer?
19:24 - 19:35: Oh, so blocking buffer is essential because once you run your gel, you have then separated out all
19:35 - 19:41: your proteins in lanes, and then you transfer that onto a membrane. And if you actually think
19:41 - 19:48: about the membrane, what it will look like is, this is my beautiful drawing. You see, there’s
19:48 - 19:54: a camera. You’ve run your proteins down the gel, and then you’ve transferred. So now we have a
19:54 - 20:02: membrane with lanes with proteins of different sizes. Now, where there’s no protein, we will
20:02 - 20:10: basically have a piece of paper that is ready to absorb stuff. So the problem you have, if I put
20:10 - 20:16: an antibody on an unblocked membrane, that antibody would just attach anywhere. It’s just
20:16 - 20:23: going to be soaked onto the membrane. So I need to block all the areas of that membrane with
20:23 - 20:31: something so that I don’t get any unspecific adherence. So your blocker will basically
20:32 - 20:38: cover everything that’s not covered by your proteins that you’ve loaded. So for example,
20:39 - 20:48: if you do a protein stain on your membrane after having transferred it, you will see little
20:48 - 20:55: red lines, almost as pretty as the one I drew, but with a blue pen. But if you do that
20:56 - 21:01: Ponseau S staining after blocking, everything will be red. And it just shows you that actually now
21:01 - 21:05: we have proteins covering everything. So yes, blocking is essential.
21:06 - 21:11: Excellent. Thank you very much, Hannah. Okay. So Karen would like to know,
21:12 - 21:16: would it be recommended to add SDS in the transfer buffer?
21:18 - 21:29: Yes. That’s a great question. So there are obviously SDS would add charge basically to
21:29 - 21:36: whatever you do. I have worked with some larger proteins. So for bigger proteins,
21:36 - 21:43: we sometimes recommend adding a little bit of SDS to the transfer buffer to, I don’t know,
21:43 - 21:47: if it’s sort of boost the proteins across, make them more transferable, give them more charge,
21:47 - 21:55: make them migrate better. So you can do that. Generally,
21:57 - 22:05: we don’t. For our normal size proteins, we rely on our general standard transfer buffer,
22:05 - 22:14: which I don’t think contains any SDS. So you shouldn’t need to, unless you are actually
22:14 - 22:22: working on larger proteins and if you do struggle with it. So not necessary.
22:25 - 22:29: Okay. Thank you very much, Hannah. Okay. Mario is asking another question.
22:30 - 22:36: Mario would like to know when staining with Ponseau S after blotting, what is the quickest
22:36 - 22:42: and cleanest solution to get rid of the stain? Water or BST or TBST? Thank you.
22:43 - 22:48: And by the way, if someone is here that asked the question, realizing that they want to
22:48 - 23:00: add additional answers, feel free to put comments in there as well. So I actually don’t really know
23:02 - 23:09: how different they are. In the past, I used to use water for doing it, but it may be that
23:10 - 23:17: the buffers would work as well. But I have one little, yeah, I seem to remember one little piece
23:17 - 23:27: of advice I was given. And I used to receive those enzymes in a little metal jar, which had a bit of
23:27 - 23:33: a sponge in it. So a tiny little sponge. And I remember putting that into the tray and it helps
23:33 - 23:40: absorb the dye quicker, but that went for Ponseau Red as well for Coomassie. So you may,
23:40 - 23:44: if you want to do it quicker, you can see if you’ve got any clean sponges lying around in your
23:44 - 23:54: lab. But yes, I’ve done water, but I’ve never really compared the water, PBST or TBST. However,
23:54 - 24:00: if anyone else on this chat line has the answer, please share with me and us.
24:01 - 24:09: Wonderful. Thank you, Hannah. Okay. Thulishitha, so again, sorry if I pronounced your name wrong
24:09 - 24:17: there, would like to know, is it worth adding 0.1% tween to the blocking buffer for the blocking step?
24:19 - 24:28: That’s a really good question. I’ve looked into this and there are
24:29 - 24:38: some people who advise not to have any tween in that blocking step. Whereas other
24:40 - 24:48: scientists, they don’t seem to see much of a difference. So in our laboratories here,
24:48 - 24:55: because we are doing high throughput and we want to make things happen quickly and consistently,
24:55 - 25:01: we actually utilize the same blocking buffer as we use for the antibody incubation. So we
25:01 - 25:09: include the tween in there. So for us, we haven’t seen much of a, it doesn’t seem to harm the
25:09 - 25:15: blocking solution, but you may very well hear that other scientists prefer not having it in there.
25:15 - 25:24: But yeah, we are currently using the 0.1% TBST with milk for our blockings.
25:25 - 25:31: I hope that helps. Excellent. Thank you, Hannah. Another question from Shajadl,
25:32 - 25:38: does stripping hamper the intensity or can stripping hamper the intensity of protein detection?
25:40 - 25:46: Sometimes after stripping, I did not get any band. What could be the problem? I checked the antibody
25:46 - 25:53: in a new membrane and it was perfectly all right. What are your thoughts on that? Well, stripping the
25:53 - 25:59: membrane, you kind of need to think about the protein. What are you doing? You are actually
25:59 - 26:06: not treating that membrane particularly well. So you’re putting on, I think some people use
26:06 - 26:15: heat, some people use really a lot of detergent to lift off the antibody to then be able to
26:15 - 26:22: reprobe it. It is a harsh environment for the membrane and I’m not surprised. We’ve all seen
26:22 - 26:29: it that it’s commonly known that you will, it’s unlikely that your protein will feel great
26:29 - 26:37: afterwards. So I’m not surprised. But what I would recommend is seeing if there are slightly gentler
26:38 - 26:45: stripping recipes that you could potentially look into to see whether there’s a way of actually
26:45 - 26:50: retaining a healthy looking protein or more of a healthy looking protein than the one you’ve got.
26:50 - 26:56: Similarly, there are commercially available stripping buffers that may work better in your
26:56 - 27:05: hands. So I would recommend doing a few comparisons if you can and see which process, which protocol
27:05 - 27:15: would work better for you. So unfortunately, or fortunately, I can’t tell you because it’s
27:15 - 27:23: also protein dependent and antibody dependent probably what is the optimal stripping condition
27:23 - 27:29: for you. But do check around or see if there’s an alternative to stripping your membranes,
27:29 - 27:36: whether it’s worth running a couple of membranes or multiplexing your antibodies
27:36 - 27:41: so you don’t have to go through the stripping step. Brilliant advice. Thank you very much,
27:41 - 27:47: Hannah. The questions are coming thick and fast now. And actually, we’ve had a reply to a previous
27:47 - 27:53: question about what’s the best buffer to use for stripping the Ponseau S. Great.
27:55 - 28:02: I found that adding PBST helps with the dye removal faster. Oh, excellent. That’s brilliant.
28:02 - 28:09: I’m going to put that into today’s learning. Thank you so much. Wonderful. Wonderful.
28:10 - 28:19: Okay, Dr. Anonymous would like to know, how can I detect the pore size of a self-made
28:19 - 28:25: polyacrylamide gel? Is that something you’ve ever thought about at Abcam or do we usually buy all
28:25 - 28:33: our own gels there, Hannah? Oh, I’m going to make you so jealous, Dr. Anonymous, in that I haven’t
28:33 - 28:40: poured my own gel for about seven years. And even when I did, I never looked at the pore sizes. So
28:40 - 28:49: I’m unfortunately the wrong person to ask this. But then again, I probably have a panel of experts
28:49 - 28:56: with us here, as I believe that we’ve got quite a number of attendees. So if any one of you know
28:56 - 29:03: how to determine pore size, please inform us. I can also make a note of that and see if I can
29:03 - 29:11: get back to you. I can speak to our protein experts. But yes, unfortunately, I don’t know.
29:13 - 29:17: Okay. Thank you, Hannah. Thank you.
29:17 - 29:24: So Sejadl also would like to know, if I detect my desired protein by ELISA,
29:25 - 29:30: in that case, if it is detected by ELISA, is it required to detect by Western Blotting?
29:30 - 29:38: What are the basic differences between these two? Well, they have something in common,
29:39 - 29:48: and that’s antibodies. So if you are interested in seeing whether your protein is in your sample,
29:49 - 29:55: they kind of give you the same readout in that, yes, you get a signal if the protein is there,
29:55 - 30:02: or you don’t get a signal, depending on that your antibodies, of course, are specific. So unless
30:02 - 30:12: you are very interested in the size of your proteins, ELISA should suffice in being able
30:12 - 30:20: to tell you whether your protein of interest is in the sample. You also have that additional level
30:20 - 30:31: of control within ELISA. You’re actually reliant on two antibodies. So I would think,
30:31 - 30:35: well, I believe that if you actually can see it with two, you’re dependent on having two
30:35 - 30:40: antibodies binding to that protein. To me, that’s even more, it tells you even more than actually
30:40 - 30:46: detecting a protein with one antibody as the Western Blot would allow. So yeah, if you’re
30:46 - 30:54: successful with ELISA, that’s great. Brilliant. Thank you, Hannah. Okay. So Anastasia,
30:54 - 31:02: another question. Is it a good idea to use BCA analysis when doing Western Blotting?
31:03 - 31:09: So BCA analysis, if I understand the question right, this is to determine the protein
31:10 - 31:18: concentration, basically. At least that is how I interpret the acronym. And this is to know how
31:18 - 31:25: much protein you have in the sample. If I’m actually answering the wrong question, Anastasia,
31:25 - 31:33: or if I misunderstand, you can just put another question in or correct me. So yeah, it’s to
31:33 - 31:39: determine how much protein you have in your sample so that you can standardize. I’m just
31:39 - 31:45: seeing if that’s, to standardize, you know how much you are putting into each lane. So yes,
31:45 - 31:52: I think it’s a very good way of determining your protein concentration. And this is what we do
31:52 - 31:59: here all the time. And as you can see from our website, we tend to standardize the protein
31:59 - 32:08: concentration and load somewhere between five to 40 micrograms per lane when we check that. So yes,
32:08 - 32:14: it’s a really good way of starting your experiment. Excellent. Thank you very much
32:14 - 32:22: indeed, Hannah. Really good answer. Thank you. Okay. Heber, again, I apologize for the pronunciation
32:22 - 32:28: there, would like to know, so I’m trying to detect a protein of about 140 kilodaltons.
32:29 - 32:36: I always have non-homogeneous transfer of this protein between different wells.
32:36 - 32:38: How can I improve the transfer?
32:41 - 32:49: Yes. So the problem is, because it’s not particularly ridiculously big proteins,
32:49 - 32:55: there’s not really an issue with the size of that protein because that’s your standard size.
32:56 - 33:04: I would look at your transfer piece of equipment and for me, it sounds as if it could either be
33:04 - 33:11: that there is something not right with the transfer. It’d be interesting to find out whether,
33:13 - 33:18: I mean, the transfer itself, if you do wet transfer, some people are worried about the
33:18 - 33:24: temperature rising, et cetera, but for a one-hour transfer, it normally shouldn’t really
33:25 - 33:31: affect it too much. But it may be worthwhile having a look and making sure that it doesn’t
33:31 - 33:38: overheat and checking that your buffers are correctly prepared and you’ve got all the
33:38 - 33:45: components in there that you’d need. It’s interesting that it seems, I’m not sure whether
33:45 - 33:53: you succeed in other sizes for the transfer, but this is where I would go. The other thing I could
33:53 - 34:01: think of is if we have, or if you have made a homemade gel and that there is some discrepancy
34:01 - 34:10: between either the thickness of the gel, which is unlikely, but whether there is polymerized
34:10 - 34:15: differently. So it could be that that would prevent transferring some regions of that gel.
34:16 - 34:25: But yes, I would go back and have a look at your transfer and see whether there’s something you
34:25 - 34:32: can do there, maybe even playing around with the time, make it fresh buffer and start again.
34:32 - 34:37: It would also probably help having a look by doing, like I mentioned, Ponseau S, where you
34:37 - 34:45: can stain the membrane to see, is it a particular region of the gel that doesn’t get transferred?
34:46 - 34:53: If so, one way to check it is actually put the gel upside down, meaning that you get to see
34:53 - 34:58: whether it’s actually a machine problem or a gel problem. If there’s one region of the machine that
34:58 - 35:07: doesn’t transfer, or is it the part of the gel that is behaving badly? Unfortunately, I would
35:07 - 35:13: recommend going back to scratch and check all the reagents to see whether that would help.
35:13 - 35:15: So yeah, hopefully that will work out.
35:15 - 35:22: Good tips, terrific. Thank you, Hannah. Okay, so this is a good question here from Shajadl.
35:23 - 35:32: Okay, so why do protein bands shift from conventional molecular weight? For example,
35:32 - 35:39: some papers show that E-cadherin might be 135 kilodaltons, although in general it is 120.
35:40 - 35:47: Why does this happen? Is it dependent on the cell, different isoforms or others? So in other words,
35:47 - 35:53: why does the molecular weight sometimes come out differently to what is reported or expected?
35:54 - 36:01: That’s fantastic. So for example, this is something that we see all of the time,
36:02 - 36:11: and this is biology. We love a bit of biology, but it doesn’t make our lives as scientists easier.
36:12 - 36:14: We have the same problems in IHC and ICC.
36:14 - 36:14: Yes.
36:16 - 36:17: We didn’t expect that.
36:17 - 36:23: No, but if you think about it, my go-to answer is post-translational modification,
36:24 - 36:34: and because often we are given, if we go to Protein Atlas and some of these other resources,
36:34 - 36:41: they often give us an expected molecular weight, which often is based on the amino acid multiplied
36:41 - 36:48: by the weight of the amino acid and then added up, and it gives you the theoretical molecular weight.
36:48 - 36:57: If we think about post-translational modification, when I think of size disparities,
36:57 - 37:03: I tend to look at glycosylation. Sugars are heavy. Sugars are extremely heavy,
37:03 - 37:11: and I think I used to work in the HIV field before, and those envelopes of the virus,
37:12 - 37:19: their molecular weight is mainly just at least 50% of them comes from all the sugars that are
37:19 - 37:26: sitting on it. In general, that would explain it. There can be differences, I believe, between
37:26 - 37:34: cells and how they glycosylate, but normally, if you get an increase in molecular weight,
37:34 - 37:41: it’s most likely due to glycosylation. There are a lot of other modifications as well,
37:41 - 37:46: which can, to a certain degree, affect your molecular weight. However, if you think about
37:46 - 37:53: the phosphorylation of a protein and the size of that phosphor group, it’s kind of minute,
37:53 - 37:58: so it doesn’t significantly shift it unless you have a lot of phosphor groups on it.
38:02 - 38:08: We have post-translational cleavage as well, where you can expect that actually,
38:09 - 38:15: upon triggering of apoptosis, your caspases get cleaved in many ways, for example,
38:17 - 38:21: so your protein can be seen at different sizes because we’ve had that post-translational
38:22 - 38:30: cleavage that happens. Talking about isoforms, this is also something that you can see that
38:30 - 38:37: there are some of the exons maybe missing in some of the isoforms. Therefore, we can see different
38:37 - 38:42: bands and depending on where your antibody is sitting, if you happen to have an antibody against
38:42 - 38:50: that part of the protein where the exon has been taken out, you may not even see the protein of
38:50 - 38:57: interest in that particular isoform. Yes, I would definitely believe that
38:57 - 39:04: post-translational modification is the main culprit here. What is also challenging is
39:05 - 39:09: comparing experiments between different laboratories and this is one of the
39:09 - 39:15: big challenges we have everywhere. One of the problems is the reporting.
39:17 - 39:23: So many times I look at journals and it breaks my heart when I see beautiful Western blots,
39:23 - 39:28: but they’ve been cropped. I see a little bit of white and I see a little bit of black bands and
39:28 - 39:34: then someone claims that this runs at 130, but I don’t see the entire gel and I don’t see the
39:34 - 39:41: ladder. Is that how they interpret what 130 looks like? Maybe it’s actually 140 or 120.
39:45 - 39:51: Please, next time you’re going to publish something really spectacular, do include as
39:51 - 39:57: much information as you can because it can help each other. It helps the scientific community
39:57 - 40:02: to understand what the molecular weight really should be. I just realized that I just went on
40:02 - 40:13: a tangent, but yes, let’s report the data properly and yes, PTM, post-translational modification.
40:14 - 40:18: Some excellent answers there, Hannah. Yeah, no worries about the tangent,
40:18 - 40:24: it’s all brilliant information. I’m sure everyone agrees with that. Awesome, let’s continue.
40:24 - 40:28: Okay, so Dr. Anonymous again, very popular person today.
40:29 - 40:35: Is it advised to use the same nitrocellulose membrane with transferred proteins twice?
40:40 - 40:46: So what you’re asking is that you’ve transferred your proteins and you’ve incubated with the
40:46 - 40:51: antibody and then you thought, oh, actually I want to probably overlay it with another antibody.
40:51 - 40:53: That’s how I interpret that question.
40:57 - 41:06: I mean, once the protein’s on the membrane, it’s possible that it stays there for a long
41:06 - 41:12: time. I know that you can dry the membrane and reprobe them or probe them later.
41:13 - 41:21: Yes, I wouldn’t recommend it actually because a lot of things will happen, particularly if
41:21 - 41:27: you can imagine that you’ve incubated your primary first round with primary antibodies,
41:27 - 41:32: secondary antibodies, you’ve incubated overnight at four degrees, it’s been sitting at room
41:32 - 41:39: temperature for hours on end, and then you’re going to repeat that. It’s not the best of
41:39 - 41:44: practices. However, if you realize that you quickly want to check something, is it there,
41:44 - 41:52: isn’t it there, it can be done. But if you have an opportunity to not do so, I would
41:52 - 41:55: recommend to not reuse an already probed membrane.
41:58 - 42:03: Wonderful, Hannah. Thank you. A lot of great questions coming in now. Okay.
42:04 - 42:14: So, another question from Mario. So, why do histidine 6HIS tagged proteins
42:14 - 42:20: migrate often faster than untagged counterparts despite having a higher molecular weight?
42:20 - 42:21: Thank you.
42:21 - 42:33: I actually haven’t seen that before. Interesting. Yeah, I have to say that I don’t know that one.
42:34 - 42:39: But if you wanted to, please, Mario, if you wanted to send us an example,
42:41 - 42:45: I’d have a look because I haven’t really reflected over that.
42:45 - 42:52: I would, yeah, I mean, my first suggestion, but it doesn’t make sense that it’s having a little
42:52 - 42:55: bit of additional charge, but migrate faster, but I don’t think it should
42:57 - 43:01: counterweight the original size significantly.
43:01 - 43:03: I was going to say charge was my additional thought, but…
43:03 - 43:10: Yeah, but it’s small. It’s so small, and that it would be detectable. So, it would be interesting
43:10 - 43:18: to see your data. If it’s a significant difference, then you do wonder what has happened with your,
43:19 - 43:26: how that construct was made, if there is something that was taken out in the cloning of it,
43:27 - 43:34: because it’s unlikely that you would see a notable difference. But yes, please share the data with us
43:34 - 43:38: and we can have a look. I hope that’s okay, Mario. And so, thank you.
43:38 - 43:41: I hope that’s okay, Mario. And sorry that I couldn’t answer that.
43:42 - 43:45: Okay. No, we’re not going to have the answer for everything, right? And it’s, you know,
43:45 - 43:51: sometimes research things, that’s science. So, yeah, that’s fantastic. Thank you, Hannah.
43:52 - 44:01: Okay. So, Balashitha would also like to know, any suggestions or advice based on your experience
44:01 - 44:05: for washing membranes in terms of duration and the number of washes?
44:06 - 44:18: Oh, yes. I’ve got a lot of advice on that. I think what we do is quickly take your, well,
44:18 - 44:25: you’ve got your membrane, it’s been sitting with the antibody, and we tend to just get rid of the
44:25 - 44:32: antibody. And then we give it a quick rinse. And you can imagine, I mean, if you’ve got,
44:32 - 44:36: if it’s covered in milk and antibody solution, it’s probably a good idea,
44:36 - 44:41: rather than wasting your time and washing it when it’s diluted a little bit. No point. So,
44:41 - 44:47: give it a quick rinse, shakey, shakey, shakey, and chuck out the buffer. Then you put your
44:48 - 44:59: washing buffer on. And we tend to do, I think it’s two times 15 minutes. And then another,
44:59 - 45:03: no, sorry, I think it’s two times 15 minutes plus another two times 10 minutes,
45:03 - 45:11: something like that. So, we have a proper exchange of buffer. I’m sure that if someone
45:11 - 45:17: really wanted to, they could probably get the same cleanliness by not exchanging buffer,
45:17 - 45:22: but putting one liter of buffer in, as long as you dilute it, but that’s probably very impractical.
45:23 - 45:31: So, I think we do in total something like five exchanges of buffers when we wash.
45:31 - 45:37: I also believe that if you go back to the webinar, there’s a little, it’s a video there on how to do
45:37 - 45:44: the Western Blot. So, we can see how many times and how we do that removal of the buffer and the
45:44 - 45:50: quick rinse. So, please have a look, but start with a quick rinse is my tip, and four to five
45:50 - 45:56: exchanges of buffer. I think give it a total between 45 to an hour of total washes, and
45:56 - 46:02: hopefully you’ll have a very clean blot. And also, sorry, and I’m just jumping back. Obviously,
46:03 - 46:11: the volume matters. So, if you have a 10 milliliter antibody solution, there’s no point
46:11 - 46:17: in washing in one milliliter wash buffer. So, I would exceed the match or at least exceed
46:17 - 46:23: the antibody volume you’ve had, so you have enough cleaning buffer to go around.
46:25 - 46:34: Wonderful. Thank you, Hannah. Great tips there. Thank you. Okay. So, Siruthi would like to know,
46:34 - 46:40: how long would you recommend storing samples for detecting phosphorylated proteins,
46:40 - 46:44: or would it be better to run them in the same day as the lysate preparation?
46:45 - 46:53: Great question. And I’m sure we have some other phosphor experts out there. If you can,
46:53 - 46:58: please get them on the gel, get them on the gel. And I think it’s very important as well,
46:58 - 47:06: when you prep your lysates, to keep it cold, keep it cold, and use inhibitors for the phosphates,
47:07 - 47:17: or phosphatases, so that you have the maximum chance of retaining those phosphor groups onto
47:17 - 47:26: the protein. And so, yes, ideally, try to do it as quickly as you can and keep it on ice if you can.
47:28 - 47:36: And yes, these are challenging modifications to look at, but yeah, try to keep it as clean as you
47:36 - 47:44: can with phosphatase inhibitors and work. And yeah, ideally, if you can avoid, don’t store it,
47:44 - 47:53: or refreeze it and defrost it. But I would also understand, you’ve got your lysate and it’s five
47:53 - 48:00: o’clock at night and you can’t, but then if possible, I would freeze it and keep it at minus
48:00 - 48:09: 80. And, but yeah. Thanks. Wonderful. Thank you very much, Hannah. Okay. Another question from
48:09 - 48:18: Shajadl. So, why is the band smiled during electrophoresis and SDS-PAGE? And what
48:18 - 48:24: guidelines would be best to prevent it? Yes, smiled bands, Hannah.
48:25 - 48:34: We always, we like smiles, but not on gels. Okay. So, some people believe or have noticed that
48:34 - 48:42: if your voltage is a bit too high, it may cause a bit of a smiling effect. And so,
48:43 - 48:50: and similarly, it could be something due to the salt concentration. And whether this is,
48:51 - 48:56: if you don’t have enough salt or too much salt in there. So, I would recommend if you could,
48:56 - 49:02: to possibly dilute the sample a bit and see whether a diluted sample in a proper loading
49:02 - 49:11: buffer would help. It’ll reduce the migration current and see whether that will let your
49:11 - 49:20: proteins or the gel run smoother. And, but yes, this is a challenge for a lot of people.
49:21 - 49:27: So, adjust the current and it could be that there’s something in your buffer that’s not
49:27 - 49:34: really compatible with the migration, whether you have excluded some of the salt or you have
49:34 - 49:40: too much salt in, it’s worth exploring. Superb. This is very educational for me
49:40 - 49:43: because I’m not a Western Blotting person. Well, you are now.
49:44 - 49:48: I’ve never done a Western blot. So, it’s incredibly educational. Thank you, Hannah.
49:48 - 49:51: There we go. Smiled bands, that’s made me smile.
49:51 - 49:54: Yes, it’s wonderful, but we don’t want that.
49:56 - 50:00: Anastasia is asking, can I overblock a Western? Is that possible?
50:04 - 50:12: That’s a good question. I’m not, I think you are very dependent on, well, you are dependent on the
50:12 - 50:19: quality of your primary antibody. And this actually links back to one of the answers Simon
50:19 - 50:27: gave a couple of days ago, that I believe that if you do, if you add 50% of milk powder,
50:27 - 50:34: you would knacker everything. You would have like a slurry of proteins sitting everywhere.
50:34 - 50:42: So, I think you can, I believe you can overblock it. But it also goes back to the choice of
50:42 - 50:49: blocking buffer, because I have a lot of examples where I’ve realized that actually using milk blot,
50:49 - 50:57: which is quite a heavy blot, actually give us relatively, in some cases have given us quite
50:58 - 51:04: weak signal on our Western blot, possibly because the concentration of the protein of interest,
51:04 - 51:12: or the amount of protein of interest is low, or the antibody is not as strong a binder as one of
51:12 - 51:19: the others. So, what we have done is to not overblock. We’ve just changed blocking buffer
51:19 - 51:26: and possibly gone for something that is a little bit lighter. So, BSA, I find blocks less of the
51:26 - 51:31: signal than milk. And similarly, we have some non-mammalian blocking buffers that we are using
51:31 - 51:37: for a lot of our infrared fluorescent Western blotting that worked really well. So, yes, you can
51:37 - 51:47: overblock. And also, but yes, so if you do realize that you’re not getting the signal that you want,
51:47 - 51:55: please look at your blocking buffer and consider that as an option to optimize. And we tend to
51:55 - 52:03: block for about half an hour to an hour before we put on primary conjugates. We have blocked
52:03 - 52:08: overnight because, you know, sometimes things happen and you’re very late in the day. And they
52:08 - 52:15: have, I don’t think we’ve had that many much issues in reduced signal, but it’s very dependent
52:15 - 52:21: on your primary antibody. So, do look around and see what else you’ve got in the lab if you worry
52:21 - 52:31: that it’s overly blocked. Okay. Thank you, Hannah. So, Jade would like to know, is it mandatory
52:31 - 52:36: to analyze the intensity of protein bands by densitometry or other software?
52:37 - 52:44: Well, you know, what did people do when I did my PhD? We may not, we didn’t have that,
52:44 - 52:51: and, or we probably did, but I, what is your, I think you need to know, what is your
52:51 - 52:59: scientific question? What are you interested in? Is it to determine whether
53:01 - 53:07: this protein is expressed in tissue A, but not in tissue B? So, that yes and no answer,
53:07 - 53:16: you don’t necessarily need to quantify protein if you can see the lack of. However,
53:16 - 53:22: if you actually want to do it semi-quantitatively, you definitely need to have, utilize the software
53:22 - 53:33: to help you determine the strength of the signal. And so, for a lot of the work I do,
53:33 - 53:39: where I have a knockout cell line, for example, I’m expecting there not to be a signal for the
53:39 - 53:44: antibody. However, if I do see a signal in that lane, I don’t need to go and quantify that. I
53:44 - 53:53: see a signal, I don’t like it. And so, I don’t necessarily use that, use software all the time
53:53 - 54:01: to determine whether my antibodies work or not. But yes, so, it’s, if you are interested in
54:01 - 54:09: semi-quantitative analysis, yes, please do, please do. It never hurts and it’s a good
54:11 - 54:16: thing to do if you have the ability. But yes, it’s up to your research questions.
54:16 - 54:19: So, in other words, Hannah, it’s like any kind of immunoassay, it’s what
54:20 - 54:25: question I’m trying to answer. Is this necessary? What do I need to do to get my answer?
54:25 - 54:32: Yeah, absolutely, yeah. Fantastic. Okay. So, Anastasia again, very
54:32 - 54:38: active today. How long can I keep a Western blot membrane? How long can I store it?
54:38 - 54:44: How long can you store it? Anastasia, by the way, thank you for being so enthusiastic.
54:44 - 54:53: And so, this is a great question. And I think if you look in the literature or guidelines,
54:53 - 55:02: you can store it for a very long time. And so, most people, they use PVDF or nitrocellulose.
55:02 - 55:07: And actually, I don’t know if there are any other membranes. These are the ones I’ve been using.
55:08 - 55:12: And both of these could be stored for a very long time. This means that you can transfer your
55:12 - 55:20: protein and then actually store that membrane. And because the protein is stuck, it sits there.
55:20 - 55:30: And I’ve been told that it stores for a long period of time. I don’t actually know because
55:30 - 55:35: I have not done a side-by-side testing myself, but I have fished out membranes that we’ve had
55:35 - 55:44: sitting for a couple of years and successfully reprobed it. The advantage of the PVDF is that
55:44 - 55:51: it’s a little bit more rugged. So, if you store your nitrocellulose and someone moves things
55:51 - 55:58: around and pulls it, it will crack easier than your PVDF. But my experience is that,
55:59 - 56:04: well, the experience I have, I haven’t tried beyond a couple of years. I haven’t done it
56:04 - 56:11: often, but I was successful in those experiments. But I think most people, they run the gel and
56:12 - 56:20: possibly want to probe within a week or so, and that’s perfectly fine. I would try them.
56:22 - 56:26: Thank you. So, I think we’ve probably got time for one, maybe two more questions.
56:28 - 56:36: So, Shajadl would like to know, sometimes after ECL, I did not get any bands. If I
56:36 - 56:42: increase the time for ECL, is there any possibility of damage of the PVDF membrane
56:42 - 56:47: since the PVDF membrane should never be dried in any steps of Western blot?
56:48 - 56:58: So, your PVDF membrane is, so you add your reagent onto it. And so, since you’re not
56:58 - 57:09: taking the reagent off either way, I can’t see that having it exposed for 20 minutes would harm
57:09 - 57:16: it than having it exposed for two minutes and not washing off the ECL reagent. So, there shouldn’t
57:16 - 57:23: be a problem with it. But I think what you’re worried about is that your PVDF membrane would
57:23 - 57:31: dry whilst after having been exposed with ECL and you got either your film on or your image
57:32 - 57:39: capture. I don’t think that should be a problem because after all, the signal is happening on the
57:39 - 57:47: membrane. And I believe that the reason we are given that advice or the advice is that during
57:47 - 57:51: your Western blot, you shouldn’t get the membrane dried. I think it’s because it could cause
57:51 - 57:58: antibodies to stick to places it shouldn’t stick. But once you’ve done your blot, I cannot see that
57:59 - 58:06: the ECL exposure for a long period of time with a potential drying up of the membrane would give
58:06 - 58:13: any additional signal because you got the enzyme in one place on that membrane. However, do correct
58:13 - 58:19: me if I’m wrong, if anyone else is online and actually have advice on that. But I don’t think
58:19 - 58:26: that should be a worry. Okay. And how do you store your PVDF membranes, Hannah?
58:27 - 58:31: So, to start with, I actually work with nitrocellulose membranes mostly. But I’ve
58:31 - 58:38: got a lot of colleagues working with PVDF membranes. And they’re stored dry. So, you
58:38 - 58:46: transfer it and store them. And my colleagues store them in the refrigerator. I don’t think
58:46 - 58:53: it’s necessary because I think once the protein’s attached to the membrane, that should be fine. But
58:53 - 58:58: yes, since my colleagues have been very successful and they have stored it in the fridge, I would
58:58 - 59:04: probably, I don’t want to challenge them. So, I keep them dry. Might as well store them in the
59:04 - 59:10: fridge if you have the space for it. Wonderful. And Hannah, thank you so much. You’ve been awesome.
59:10 - 59:17: That brings us to the end of this Western Blotting Q&A conversation. Thank you very much
59:17 - 59:22: to everyone who sent questions in and listened to this session. Really appreciate you joining
59:22 - 59:32: us and staying with us and sending those questions in. That’s fantastic.

About the presenter:

Hanna Dreja received a Ph.D. from Imperial College (London) and completed postdoctoral research at several academic institutes (Institut de Génétique Moléculaire de Montpellier (France), Queen Mary’s University (London) and the University of Cambridge). She has developed a broad scientific skill base with a strong focus on antibodies and infectious agents.

As a team leader of the Scientific Quality Control team at Abcam plc., she works on a mission to provide products that exceed our customers' high expectations - helping them to discover more, faster. The knockout initiative has been a cornerstone of the recent successes of the team and was recognized in the prestigious CiteAb award in 2016 and 2020.

Western blotting at 40

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