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Review this popular and powerful method for accurate detection of low abundance proteins or changes in protein expression.
This webinar is intended for scientists who would like to learn about and incorporate more sensitive fluorescent detection methods into their western blot protocols.
Dr. Martin Broadstock is currently an Edmond and Lily Safra Research Fellow at King's College, London.
He has worked in biomedical research for the last ten years, where he investigates new therapies for the treatment of Parkinson's Disease dementia.
Joining Martin today will be Augustine Mzumara, a member of our new products team. Augustine has an MSc in Human Molecular Genetics from Imperial College, London. Before joining Abcam, Augustine has worked as a research scientist at GlaxoSmithKline and has a diverse background in molecular biology. When you log-off from this webinar, you will be redirected to a webpage where a copy of the presentation site can be found and downloaded. I will now handover to Martin who will start this webinar.
So, really, my kind of research looks at synaptic dysfunction in a wide range of neurodegenerative diseases; and looking at therapies and their potential effects on these dysfunctions. Technique-wise, I routinely perform westerns all of which are now fluorescence. I do immunohistochemistry and immunocytochemistry, and most of which are now fluorescence. I also do some in vivo work, which I'm not going to go into today. So just to highlight, at the Wolfson Centre we've actually now got rid of our dark room within the building, and we've all been converted to the joys of fluorescent western blots. Just as an example image - here's on the right is an example image, which we've converted to greyscale for ease of publication. These are multiplexed westerns whereby you've got two images from the same gel using antibodies, mostly from Abcam, which show alterations in synaptic protein expression following a pro-oxidant diet treatment in an Alzheimer's mouse model.
So now we're going to talk about fluorescence and how it compares to ECL, which many people are familiar with. There isn't much difference with ECL and fluorescence in terms of the initial stages of running a western. Though you start off as you would normally by extracting your proteins from your tissues or your cells, quantifying your protein total cell load, which I've not shown here. Once you've either diluted all your samples or your same concentration per volume, or worked out your volume to load onto your gel, you load and you run your gel. You then transfer the gel to a membrane, typically either PVDF or nitrocellulose, you block your non-specific binding and then you incubate with your chosen primary antibody. Then after a while you incubate with your secondary antibody, and here's where the two different detection methods start to differ.
So using horseradish peroxidase conjugated antibodies and ECL, you have to setup your ECL detection reagents. You then expose your gel to film and then finally you can analyze your image and you get, hopefully, something nice and clear like is shown on the top left. With fluorescent western blotting, however, you can go direct from your antibody staining straight to the image analysis. You can see already that this is streamlining the workflow somewhat, and really allowing you to get on with other things that you've got on in the lab.
What are the advantages of doing fluorescent western blotting? Well, one is the improved signal that you can see compared with HRP and ECL. With ECL the signal's generally accepted to be stable for about half an hour, once you've mixed everything and applied it to the membrane. With fluorescence, however, the signal is much more stable; indeed, I've unwrapped some membrane and rescanned this a year later and got exactly the same results in terms of quantification. The membranes can be archived for projects which are ongoing in the lab, and may require independent verification later on. ECL can also sometimes result in smaller weight proteins being obscured, whereas fluorescence allows these smaller proteins to be more accurately resolved. Furthermore, fluorescence generally shows an improved sensitivity, so they're able to detect much smaller quantities of proteins when compared with ECL. So there are sensitivity issues, but also, and quite important in today's funding climate, reduce cost with fluorescence.
Whilst it’s true that the initial outlay for some of the scanners can be quite expensive, if your department's running a fair number of westerns routinely, it might actually be more cost-effective to switch to fluorescence detection. Here you can do away with the need of film and developers, which in themselves aren't inexpensive items and developer is often hazardous too. You can also do away with a need for a dark room, as most scanners nowadays are bench-top ones and can be easily incorporated into the lab. Another key saving is the cost of antibodies; I generally use secondary dilutions from around 1/10,000 to 1/20,000, which means your small pots of antibodies last for an awfully long time, and the outlay for these antibodies is often the same as a HRP conjugated one. Further savings on a blot-by-blot basis are the reduced need for molecular weight markers, many of which, even if they aren't marketed as fluorescent-suitable, will appear fluorescent once they've been scanned. Indeed, the LI-COR website suggests the cost of $1.84 per fluorescent western blot, $18.39 per ECL blot without stripping and reprobing, and $27.42 per ECL blot with stripping. So this is saving you, if you're using fluorescence, around $20 or roughly £15 per blot. So you can see if you're starting to do 100 gels in a day, you're going to rapidly make up significant cost savings.
Apart from the enhanced sensitivity and the reduced costs, the other major advantage is this ability to multiplex, or to detect two proteins simultaneously on a single membrane. For example, you may want to look at phosphorylated proteins and compare these levels against their non-phosphorylated counterparts. Though, using the old system you could either run two ECL gels or try stripping and reprobing for the different isoforms. With fluorescence you can simply run one gel and use two different primaries, and two alternatively fluorescent secondaries on the very same gel. This way you've got no problems with stripping and reprobing, and the samples have been run in exactly the same way with the same efficiency of transfer. We routinely do this for our protein of interest together with a housekeeping protein; for myself it's probably beta III tubulin or actin, and this allows normal, accurate normalization of proteins on the same gel. Stripping and reprobing can be quite tricky if your proteins are close in size, and there's always this risk of starting to remove the proteins from the membrane itself, if you're a bit over keen with your stripping. The last advantage, which is the reduced time that I eluded to earlier, which means that you can be getting on with all the other tasks that you have in hand.
So I might have started to convince you to go fluorescent, but what's the best way to make the switch from ECL to fluorescence? In the next two slides I've put together some thoughts on different issues that we encountered when we first made the switch. I'd just like to remind you to please feel free to submit any questions by using the Q&A panel on the bottom right hand corner. The first issue that you might be having is playing around with the primary antibody concentrations. Generally, you're probably going to have to reduce them, in my experience, as too much staining can almost burn out some of the scanning systems, which can result in reduced accuracy of detection. So optimize your antibody concentrations, sometimes the primary's fine but you might well have to play around with the secondary, and select those dilutions which give you a nice, clean background signal. To be honest, this is probably the most important issue when making the switch, just having to do a tiny bit of reoptimization and, generally, if I could suggest, using lower concentrations.
The other thing is it's very important to use a membrane with low autofluorescence, as otherwise you're going to be struggling with the signal-to-noise ratio in detecting and the quantification of bands. Many suppliers actually state low autofluorescence now on their membranes, and there's low fluorescent PVDF membranes available. Then have a look at your blocking buffer, so in common with ECL different blocking buffers can give you different effects, particularly on primary antibodies. I routinely use 5% skimmed milk in PBS Tween.
Whilst on the subject of buffers, make sure your chosen buffer is completely dissolved and that there's no little bits of particles in the solution. These particles can settle onto your membrane and create fluorescent artefacts, if they do so. Also, interestingly, biro markers tend to have some autofluorescent capabilities and sometimes it might happen, certainly they've smeared. So if they're currently used, maybe try and use a pencil instead. Don't press too hard on the membranes because sharp objects can sometimes leave fluorescent artefacts as well. So try and handle them with blunt forceps.
Particularly then, in terms of fluorescence, most of the primary antibodies used are relatively photostable and they won't fade within normal laboratory lighting. Although, I must admit, I tend to incubate my membranes in the dark as old habits die hard. I always, however, wrap foil around my antibody stocks and keep these in a lightproof box, just to avoid any chance of potential photobleaching.
When handling the membrane at all, always use powder-free gloves and otherwise you'll end up with a messy membrane with fluorescent fingerprints on the blot; so it's not difficult to see who's the culprit!
I tend to avoid stripping and reprobing of my fluorescent membranes, to be absolutely honest. By multiplexing I've already accounted for the total protein using an internal control. I found antibodies against very highly-abundant proteins quite difficult to remove from the membrane, without affecting the total protein from other areas of the membrane too. For lesser abundant proteins it can be done, and there are many recipes online and proprietary solutions available for a range of suppliers. Like I say, it is something that we've done before kind of lesser abundant proteins.
Lastly, if you're going to be using bromophenol blue in your loading dye, I'd suggest that you ensure your dye front has run sufficiently far away from the expected protein size, as this dye has a tendency to fluoresce. Having said that, I use this in all my samples at all times, but I found by varying the gel percentage I'm able to ensure that the dye front has passed my area of interest.
So now that I've touched on multiplexing, here are a few pointers which can hopefully help you get the most out of this exciting advance. It sounds very obvious, but it's worth pointing out that if you want to multiplex you'll need to use primaries, which are raised from different species. It's also worth considering that you might want to use primaries from species which aren't closely related - like mouse and rat - as these antibodies can have a tendency to interact with each other. I'd suggest you use secondary antibodies which have been highly cross-adsorbed, and these can prevent channels from bleeding into each other. Use fluorochromes with distinct and non-overlapping spectra. As a general rule of thumb, I'd also optimize each antibody independently of each other prior to using them together in a multiplex. This way you can validate both of your antibodies, and because sometimes antibodies will give you secondary, non-specific signals, if you're trying to multiplex and you're trying to see a specific signal, it may be that this specific signal is masked by the non-specific signal of the other antibody.
So being able to multiplex is a very useful advance in western blotting, but what do I think the future holds for fluorescence? Well, in a sense, these technologies are already around and in use today, and in the absence of a crystal ball or any other means of divining what the future holds, I thought I'd briefly mention these as they have some specific advantage over the currently used methodologies. The in gel western is exactly what it says: it's here you remove the transfer step to nitrocellulose membrane completely, and instead you stain the gel directly with antibodies. This makes it ideal for very large proteins which can often be difficult to transfer efficiently, without losing the smaller proteins onto the same gel. You remove the blocking steps and just use your primaries and secondaries directly onto the gel itself. Many people find this sensitive enough to use and, like I say, particularly for those hard-to-transfer proteins. However, it can be somewhat cumbersome dealing with the gel directly, and tears can happen by handling the gel; normally, in my experience, always in the wrong place.
Another alternative is to do away with the gel-based system completely, and this can be achieved through the use of on or in-cell westerns. Depending on where your protein is located, determines whether or not it's on or in the cell. Methodologically, this is most similar to performing immunocytochemistry where you fix your cells, permeabilize the membrane or not, if you're looking at membrane-bound proteins add your primaries, and then add your subsequent secondaries. I normally do this in combination with actin to control for cell number variations between different wells. It could make the analysis slightly more complicated, but the process itself saves an awful lot of time by doing away with the harvesting, the running gel steps; so this more than makes up for it. It can also be used to validate RNAi interference and has a high sensitivity. Essentially, if your antibodies work for immunostainings and for westerns, they may work well for both on or in-cells.
To visualize your fluorescent readouts you require, obviously, an imaging system which may be in use for a different application within your department already. The two main types of system: infrared and non-IR systems, and the IR system is the one I'm most familiar with and this is the LI-COR Odyssey system. It uses two lasers at 700 and 800 nm to excite fluorochromes in the infrared range, which LI-COR report to have very low autofluorescence, because of this infrared range. The disadvantage is that you can't generally use then your secondary antibodies for other applications, because most microscopes won't pick out anything in the infrared range; although, the on gel and in-cell westerns are fine.
Alternatively, you could use one of the non-IR systems, and there's a wide-range of these available from many suppliers. With your imaging system now in place, I thought I'd briefly review the four most common pitfalls and possible solutions to fluorescent western blotting, namely: globally high background, spotty background, weak signal and non-specific bands. Some of these issues would remain the same for both ECL or other detection systems, then that way it can be viewed as a general tool for optimizing westerns.
So here I've tried to think of possible causes to the high background, together with some technical tips and tricks to try and help eliminate those causes. High background often results from inadequate blocking, so I'd suggest this is your first place to have a look. Try using alternative blocking solutions, either BSA or milk. Try adding some detergent such as Tween 20 or SDS to remove unwanted proteins from attaching to the membrane. Optimize your antibody concentrations and, importantly, keep the membrane uniformly wet, and try and handle it as little as possible.
Another issue can be the appearance of spots within the background, rather than a global increase in background fluorescence. Again, keeping your membrane uniformly wet, cleaning the scanner and filtering any blocking solutions that you use should help to keep this issue under control.
A weak signal is often encountered with blotting, so for these issues I'd recommend increasing your total amount of protein loaded onto the gel in the first place, and then altering the primary concentration before trying any of the other steps that I've suggested. In addition, you can try altering your secondary concentration and I would always recommend using a stain after transferring, such as ponceau stain to check your efficiency of transfer, and also the appearance of the dreaded bubbles on membranes. If the transfer isn't the issue, you might want to re-examine your membrane as small proteins in particular may pass through the membranes, whilst larger proteins are only starting to migrate. I'd suggest, in this case, either reducing the transfer time or trying a smaller pore-sized membrane.
The last issue I'd like to tackle is that of non-specific bands. If looking at each antibody here independently; here the culprit is often too high a primary antibody concentration, or too long an incubation. To try to reduce the amount of primary or try incubating it for a shorter time period, if you're doing it at room temperature, or try an incubation overnight at 4°C, which I found to be my optimum. When you're multiplexing use antibodies from different, and preferably not closely related, species. Check the spectra of your different antibodies to make sure there's no bleed through, and if this doesn't work you can try reducing the protein total loaded onto your gels to reduce the signal, whilst maintaining specificity. I'm going to be hanging around to help out with any questions you might have, so please feel free to get in touch and I'll do my best to answer at the end of the session. Now I'm going to hand you over to Gus.
AM: Hello everyone. Thank you for a really interesting and informative presentation, Martin, and the very valuable troubleshooting tips for fluorescent western blotting. Now, I'm going to go into protocols and resources that might be useful for your fluorescent western blot experiments. Before I do, let me just remind you that if you have any questions, please feel free to submit these through the Q&A panel on the right-hand side of your screen.
I'd like to start by highlighting Abcam’s extensive and validated secondary antibodies for fluorescent western blotting, including our FITC, DyLight and CyDye pre-adsorbed secondary antibodies for minimal species cross-reactivity.
We are also pleased to let you know that we also now offer Alexa Fluor® 488, 555, 594 and 647 conjugated secondary antibodies. Why should you choose one of our Alex Fluor® conjugated secondary antibodies? All of our Alexa secondaries have been extensively tested in-house to guarantee bright staining and low background. As part of our QC process we do a secondary antibody control at a 1/200 dilution to ensure the specificity of the secondary. Furthermore, the selection of pre-adsorbed secondary antibodies is large to ensure low species cross-reactivity and recognition of antigens immunoglobulins. If you would like to use these products and ICC as well, you can do at least 250 experiments, assuming a 1/200 dilution. Find out more about these on the link shown.
Our catalogue also includes an extensive range of antibodies and proteins for direct fluorescent detection. The example shown shows a five-star review of the beta tubulin antibody used in a fluorescent western blot. Fluorescent conjugated primary antibodies, or detection in purification proteins can be found using our advanced search tool, and selecting the required conjugate on the left hand side of the Abcam website, as shown.
Another useful range of products for setting up your fluorescent western blots for direct detection, are the EasyLink antibody conjugation kits. EasyLink antibody conjugation kits offer a rapid and easy way to fluorescently label your primary antibodies. The range includes a choice of 18 fluorescent labels that are available in a range of various convenient sizes, to give you flexibility in experiment design. Find out more about the EasyLink range on the link shown.
Some of the benefits from EasyLink kits include they're use for direct staining protocols, where you can benefit from multiplexing and avoid cross-reactivity. The labels also covalently attach to a free aiming group, which produces a perfectly stable conjugate for your experiments. With only 30 seconds hands-on time, there are cost-effective and rapid solutions of having the primary antibody that you would need for your experiments. One thing to note about the EasyLink conjugation kits, is that conjugation is dependent on the antibody concentration. We have concentration kits available to help you with this, and ensure you have the right concentration. In addition, conjugation can also be affected by the presence of BSA and the azides in the buffer; and we also have purification kits to help you with this. Typically, for a conjugation you'd want an excess of labelled antibody, and conjugation in a ratio of 1:1 or 4:1.
Just to remind you again, if you do have any questions please feel free to submit these through the Q&A panel. To simplify your transition to performing fluorescent western blots, I'd also like to highlight the Optiblot fluorescent western blot kits. They allow the fluorescent detection of two proteins on one blot with no need to strip and reprobe. The kits contain all the reagents you would need to conveniently perform fluorescent western blots. Included with the kits are low fluorescence PVDF membranes, optimized buffers and two fluorescently labelled secondary antibodies. The secondary antibodies provided with kits are brighter than Cy dyes and can be visualized with the Cy3 green and Cy5 red channels. The kit is compatible with a number of non-IR-based fluorescent detection systems, which are shown and some of which Martin mentioned earlier on.
If a transition to fluorescent western blot is still a challenge for you, the reagents that would allow you to benefit from some of the advantages of fluorescent detection is the range of high-sensitivity Optiblot ECL substrates. In addition to being chemiluminscent, these substrates are also fluorescent allowing increased sensitivity and to maintain signal. The substrates are excited using the blue 488 nm laser, and can be detected using an orange filter compatible with the emission of a Cy3 dye. The Optiblot range of ECL kits also offer a range of other features that will enhance the sensitivity and flexibility of your western blot protocols.
In addition to reagents for fluorescent detection, within the Optiblot range you can also find a wide range of products designed to enhance and simplify your western blot protocols. This includes Alexa fluorescence gels with a two-year shelf life, and dyed wells for simpler loading. Buffers and accessories like Optiblot blue, which is a coomassie stain that offers gel staining in just 15 minutes, as well as protein ladders and low fluorescence PVDF membrane.
In addition, you can find a wide range of resources on the Abcam website, which can further help you with troubleshooting or setting up your fluorescent western blots. Please visit the links shown to find further information. There is also western blot troubleshooting tips booklets, which you can download as a valuable tool for your experiments, or alternatively you can email our customer service teams to request your own personal copy.
Lastly, I'd like to mention, as a thank you for attending the webinar we're also offering a promotional discount of 25% on the products mentioned in this webinar. Further information about this offer will be sent after the webinar.
I'd also like to take this opportunity to let you know about some of the additional events that we have coming up during 2013 that might be of interest to you. Firstly, there is the Allergy and Asthma Conference which is happening from May 23rd to May 24th in Bruges, Belgium. Please take note of any abstract and posted deadline dates. Next of all, we have our Inflammasomes in Health and Disease, which is happening from June 24th to 25th in Boston, US.
In addition to some of the conferences that are coming up, there are also some additional upcoming webinars which may be of interest. This includes “IHC/ICC staining techniques using single and multiple labels”, on 6th March; and “To interact or not to interact? Immunoprecipitate to answer this question”, which is happening on March 21st. I'd like to thank you again for your time, and for listening to the webinar. I'm now going to hand you back to Martin who will answer some of the questions that you've submitted. Thank you again.
MB: So I've got a question from Caitlin, which says: I have found in comparison that I need to load more protein per well for fluorescent detection compared to ECL, and the ECL seems to be more sensitive. Here, I think it depends pretty much on the detection system that you're using. I have certainly found ECL to be less sensitive, even the ECL advance. It may well depend on the antigen that you're looking at.
Victoria asks: Do you have any recommendations for secondary antibodies in the LI-COR system, as I am experiencing an extremely low signal compared to ECL? Victoria, I get my antibodies direct from LI-COR, but you can also use some Alexa Fluors® and I can't remember them off the top of my head, but I will be able to get back to you on that, so we can sort something out there.
Molly has asked: I'm trying to compare two separate NMDA receptor subunits, NR2A has a great signal, but NR2B barely shows up; is it okay to load different concentrations of antibody for these two subunits? Yeah, it's absolutely fine. You're using two different subunit-specific antibodies. Absolutely fine, no problem!
Molly asks again: How do you recommend I store fluorescent western blotting membranes for future imaging, if we want to? Keep them wet, keep them in the dark, so wrap them up in cling film and keep them a bit moist, and then also put them inside some tinfoil is what I do, and then stick them in a cold room.
Rahila says: If we're switching to fluorescent western blotting, we will have to use fluorescent antibodies, but can we use these fluorescent antibodies for routine immunocytochemistry? To be honest, Rahila, this will depend on the detection system that you're going to be using. You won't be able to do this with the LI-COR system, but you should be fine with any of the others. Nadine asks: What's the sensitivity for fluorescent western blots, is it pg or fg? Definitely picograms, but, again, it'll depend on the detection system that you're going to be using.
This is Lava asks: What is ECL? ECL is Enhanced Chemiluminescence.
Thank you, Martin and Gus. We've reached the end of our webinar. For any people whose questions have not been answered, we will be contacting you shortly with a response. Also, to let you know, a PDF copy of this presentation is available for download. When you log-off from the webinar, you will automatically be redirected to a webpage where the downloadable version can be found, along with information about the special webinar promotions that are running. If you have any questions about fluorescent western blotting or any scientific enquiry, please don't hesitate to contact our scientific support team who will be very happy to help you. They can be contacted by email at firstname.lastname@example.org. We hope you have found this webinar useful and informative, and we hope to welcome you to another webinar in the future. Thank you for attending and good luck with your research.