Advanced Immunoprecipitation webinar

To interact or not to interact? Immunoprecipitate to answer this question

Identifying bona fide protein-protein interactions can be a difficult task due to the complexity of protein interactions. Immunoprecipitation of proteins can elucidate the molecular importance protein-protein interactions play in signal transduction.

Join our presenters as they discuss how to identify protein interactions though immunoprecipitation and overcome biochemical problems that can be associated with this technique. 

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Webinar Topics:

  • Sample preparation
  • Positive and negative control design
  • Different binding and elution techniques
  • Upscaling of IPs
  • Validation of interactions (yeast-two hybrid/mutational analysis/mass spectrometry/western blotting)
  • Evaluation of the functional role of an interaction
  • IP hints and tips

About the Presenters:

Seán Garry

Seán Garry received his degree in genetics from Trinity College Dublin in 2006 and continued into research with a PhD in the laboratory of Dr. Margaret McGee at the Conway Institute, University College Dublin. For his thesis, Seán investigated how the pro-apoptotic Bcl-2 family member, Bim, is phosphorylated following prolonged mitotic arrest.

After his PhD, Seán relocated to the laboratory of Dr. Viji Draviam at the Department of Genetics, Cambridge University, to carry out post-doctoral research into how kinetochore-microtubule attachment is established during mitosis. 

Rachel Imoberdorf

Rachel Imoberdorf studied Biology at the University of Bern, Switzerland. She then moved to Geneva, Switzerland where she did her PhD in Molecular Biology in the laboratory of Prof. M. Strubin.

During her thesis Rachel was interested in understanding the effect of global histone acetylation on transcriptional regulation in the yeast S. cerevisiae. She also worked on the development of a system to measure transcription factor turnover on specific regions in the genome. 

Rachel is currently a principal scientist at Abcam and has set up IP in our lab, thus enabling us to routinely test our own antibodies with this technique.

Webinar Transcript:

Good day ladies and gentlemen, and thank you for standing by. Your host for today's presentation: To interact or not to interact: Immunoprecipitate to answer this question, is Sarah Dolny Event and Marketing Coordinator from Abcam. I would now like to turn the presentation over to Sarah.

SD: Thank you very much, Rick. Today's presenters will be Rachel Imoberdorf, Sean Garry and Judith Langenick, all from Abcam. Rachel gained her knowledge in IP during her PhD at University of Geneva in Switzerland. Rachel is a principal scientist at Abcam and has set up IP in our lab. This assay allows the laboratory to routinely test our own antibodies in this fantastic technique. Sean received his PhD at University College, Dublin and spent countless hours trying to IP various kinesis involved in mitotic progression. Following this, Sean transitioned to a postdoc at Cambridge University where he carried on trying to identify proteins involved in a checkpoint throughout both yeast, two-hybrid and IP. Finally, Judith will be joining us today. Judith completed her Molecular Biology degree and her PhD at University of Dundee. Upon completion of her PhD Judith moved to the MRC Laboratory of Molecular Biology in Cambridge. Judith joined Abcam in 2011 and is a Product Manager.

As a reminder all of our presenters will be receiving questions throughout this webinar, so please submit your questions on the right hand panel of your screen. Also, powerpoint slides will be emailed directly to you within the next few days. Please take a moment to share your thoughts with us about this webinar and the entire webinar series within a survey that will follow this webinar. Now, I would like to turn the presentation over to my colleague, Rachel.

RI: Welcome to our webinar. In this section I will give you a short introduction to IP, I will then talk about how to set up an IP experiment and how to find the perfect antibody. How to find the antibody to the beads, and I will talk about antibody titration and how to choose a secondary reagent for IP western blot. 

Even though this is an advanced IP webinar, I thought we would start with the question: What is IP? IP is a technique used to enrich or purify a specific protein from a complex mixture, for example, an extract using an antibody. What can you do with IP? You can isolate a protein from a complex mixture to then use in other assays. It can also be used to concentrate a protein, for instance, if your protein is expressed as very low-level, IP might be required to visualize it in western blot, or you can determine protein-protein interactions in so-called co-IP. In this technique the protein of interest is immunoprecipitated and you then check either by western blot or mass spec, if other proteins were copurified with your protein of interest.

In the next slide you can see a typical IP western blot, this is an IP we performed in our laboratory using an antibody specific for TBP, the TATA-binding protein. The bands created by TBP are highlighted by the red square. You can see a nice enrichment of TBP in the IP in lane three, compared to the extract in lane one. This blot contains all the typical IP western blot controls.

I just wanted to talk you through these IP western blot controls. The first lane is the extract or input, the starting material. This is loaded on the gel to determine if the target can be detected in the starting material. This is not always the case, as sometimes enrichment by IP is required to visualize the protein. The second lane is a fraction of the supernatant after the immunoprecipitation. This is loaded on the gel to determine if the target protein has been depleted from the extract during the immunoprecipitation, and it will tell you how efficient your immunoprecipitation was. The third lane is the immunoprecipitation, so the outcome of the experiment. If the IP was successful and efficient, this band should be enriched compared to the extract. The fourth lane is a sample where the immunoprecipitation has been performed with a lighter-type control antibody, so an antibody of the same isotype as the one used in IP, but not recognizing the target protein. This control is important as it will tell you whether the bands you see in IP are due to the antibody, or due to its specificity for the target. The last lane is the bead control where everything but the antibody was added to the reaction. It is important to include this control in your experiment, as it will tell you if there are bands in your IP that are due to non-specific binding of proteins to the beads.

After the short introduction, I would like to tell you more about how to set up an IP experiment. The two most important steps in setting up an IP experiment are to find the perfect antibody. This is absolutely key if your antibody does not work in IP, your experiments will not work. Once you've found the perfect antibody for your IP, you will need to determine the right testing conditions for your experiment. Let's start with finding the perfect antibody.

The best way to start is to check if there are any references in PubMed where an antibody has been used to IP your protein of interest by other researchers. Then if different antibody suppliers for an antibody that is tested in IP on all datasheets, for instance, this would be noted in the tested applications here. Also check if there are customer reviews for IP, these are very helpful as they often give you a good idea on how well the antibody works. On our datasheets these can be found in the AbReview section here.

If there is no IP tested antibody available, these are the things you can watch out for. In IP the target protein is usually the native conformation, therefore, an IP antibody needs to recognize an epitope on the exposed surface of the protein. Furthermore, as it is a pull down reaction, the antibody also needs to have a high affinity for the epitope. So if you look for an antibody that works in IP, it is worth checking if the immunogen is on the exposed surface of the protein. You can only do this though if the structure of the target protein and the immunogen used to generate the antibody are known. Other good indicators for success in IP are if the antibody works in ChIP or immunohistochemistry (IHC), as these techniques have similar requirements for the antibody as IP.

A question we often get is: Should I choose a poly or a monoclonal antibody? Polyclonal and monoclonal antibodies can both be excellent IP antibodies, but they have different advantages and disadvantages. Monoclonal antibodies are specific to single epitope, whereas polyclonal antibodies consist of a pool of antibodies that recognize different epitopes. The probability of the polyclonal antibody works in IP is, therefore, higher than that of a monoclonal antibody, but this does not mean that the monoclonal antibody will not work in IP. Monoclonal antibodies do have an advantage due to the nature of how they are made, they usually show minimal batch-to-batch variation. Polyclonal antibodies are more prone to show batch-to-batch variation as they need to be remade by immunizing another animal. We do extensive testing of each batch to ensure each new batch is comparable to the last one, but slight differences between batches are almost impossible to avoid. 

That's now covered how to find perfect antibodies, and we'll discuss the next step: the binding of antibodies to bead. You will first have to determine which type of beads you want to use. There are generally two types of beads available: Agarose/Sepharose beads. These have been around for a long time and are easy to use, and easy to setup. Another type of support are magnetic beads. These are also very easy to use, but you need to have the right magnet. The advantages of these beads are there is no need to centrifuge, they have short incubation times and it is easy to remove the supernatant, and they are quite easily scalable for throughput. 

Once you have decided which supports you need, you need to decide on which type of Ig binding proteins you want to use. The most commonly used Ig binding proteins are protein A and protein G, which are bacterial proteins. These proteins bind to the FC part of the antibody, with different affinities depending on the species and the isotype of the antibody. As you can see here in this table, rabbit IgGs are bound with similar affinities by both protein A and G, so either can be used for IP with rabbit antibodies. On the other hand, protein G has a higher affinity for mouse IgG1 antibodies than protein A, so you would choose protein G for an IP with this type of antibody. There are tables available to help you determine if protein A and/or G is the better choice for your antibody. A link to the table on the webpage is here below this yellow table. 

Once you have decided on the antibody, the support and the IgG binding protein, you can start setting up the experiment. One question we often get is, in what order should the components be added? Binding the antibody to the bead first and washing the antibody bead complex before adding the extract is good practice. The reason for this is that there is a protease cleavage site between the Fab and the FC part of the antibody. So if your antibody is partially degraded, you will end up with Fab fragments that are able to bind to the target protein, but that cannot bind to the beads. This means that you will lose part of the target protein from your IP reaction. If the antibody is bound to the beads first and then washed, these Fab fragments are washed off and only antibodies that can bind to beads will be added to the extract.

I have now covered how to choose the beads and will move on to antibody titration. One question we often get is how much antibody should be used in an IP reaction? The best way to determine the right amount of antibody required, is to do a titration experiment. 1-10 µg of antibody for 500 µg of extract is usually a good range. One thing to consider here is also to have a good antibody bead ratio; you don't want to use too many beads, as they can result in background and not too little, because otherwise you will lose some of the antibody. 

Another the important step in the setup of IP experiments is the choice of the secondary reagents for the western blot. The reason for this is that usually during the elution step, not only the target protein, but also the antibody is eluted from the beads. The antibody can then result in bands of 55 and 25 kDa in western blot, which are created by the heavy and light chains, respectively.

So if you use a primary antibody that is raised in the same species as the IP antibody for western blot, the secondary antibody will not only recognize the primary antibody, but also the denatured IP antibody heavy and light chains that are present on the blot. This results in large black bands on the western blot at 55 and 25 kDa, and can interfere with detection of your target protein or the protein you want to co-immunoprecipitate.

There are several options to overcome this problem. The first one is to use a primary antibody of a different species than the IP antibody in the western blot, so that the secondary antibody will recognize only the primary antibody used in western blot, and not the antibody present on the membrane that stems from the IP reaction. However, an antibody from a different species is not always available. For proteins that are bigger than 30 kDa you can also use light chain-specific secondary reagents. These antibodies will only bind to the light chains of the primary antibodies, and would therefore only result in a band at about 25 kDa on a western blot gel. You can see an example here where we did an immunoprecipitation with an antibody against HDAC2, and then used the same antibody as the primary antibody in the western blot and developed it with a light chain-specific antibody. You can see the light chain bands around 25 kDa, but there are no bands at 55 kDa in the IP. We offer a range of light chain-specific secondary antibodies on our webpage, and Judith will tell you more about them later. 

Another option is to use a conformation-specific reagent that only recognizes the antibody in the native conformation, so it will not recognize the denatured antibody on the membrane. We offer one of these reagents, the so-called VeriBlot for IP. This is an image of an IP western blot where we used VeriBlot for IP as secondary reagent, and you can see that the only band present in the IP reaction is the 17 kDa histone band of the target protein. There are no bands at 25 or 55 kDa where the light or heavy chain bands are.

So, in summary, we can say immunoprecipitation can be used to identify protein-protein interaction. For an antibody to work in IP, it usually needs to recognize an epitope on the exposed surface of the protein, and to have a high affinity for the target protein. Choose beads coated with protein A or protein G, dependent on the species and the isotype of the IP antibody. Binding antibodies to beads first is good practice, and the secondary reagent choice is key for IP western blots.

Before I hand over to my colleague, Judith, I just want to remind you that in case you've got any questions, you can submit them throughout the webinar simply by typing them in the Q&A box on the right hand side of your screen. Thank you for your attention.

JL: Hello everybody. As Rachel mentioned, it is quiz time so let's see how good your IP knowledge is. The quiz will appear in the right bottom corner of your screen, and you have two minutes to answer the three questions. Thanks for taking part. The answers will be revealed after the Q&A session. I'm now handing you back over to Sean.

SG: Thank you Rachel and Judith. For my section of the talk I will discuss RIPA and NP-40 buffer, maintenance of post-translational modifications, buffer optimization, protein overexpression, bioinformatics and confirming protein-protein interaction.

Depending on the expression of endogenous proteins produced by our cell type, identifying protein interactions may require some optimization. To begin with, immunoprecipitate from 300 µg of whole cell extract protein. However, if no interactions are observed by western blot, increasing protein in a range up to 2 mg may increase visualization of the interaction. If no pull down is observed, immunoprecipitating from a subcellular fractionation may increase the possibility of detecting your interaction. Depending on the subcellular localization of your protein, pooling isolated nuclear mitochondrial cytosolic or membrane protein fractions will increase the amount of subcellular protein, and therefore increase your chances of detecting an interaction. Today, we will not be discussing subcellular fractionation; however, numerous protocols are available online for subcellular fractionation. Here we have listed a few published protocols for the isolation of each subcellular compartment. 

Depending on what proteins you're immunoprecipitating, different cell lysis buffers may be required. Lysis of cells with RIPA buffer gives low background following IP, but can denature some kinases. It also has the potential to disrupt protein-protein interactions. RIPA buffer enables rapid, efficient lysis and solubilization of proteins from both adherent and suspension-cultured mammalian cells. RIPA is a widely used lysis and wash buffer, and it's most antibodies and protein antigens are not adversely affected by the components of this buffer. In addition, RIPA lysis buffer minimizes non-specific protein binding interactions to keep background low, while allowing the most specific interactions to occur, enabling studies of the relevant protein-protein interactions.

Once samples are ready to lyse, buffer supplements need to be added fresh to the RIPA buffer. Supplement reagents include EDTA, which is the metal collator and a phosphatase inhibitor, sodium fluoride, which is a serine/threonine phosphatase inhibitor and protease inhibitors to prevent protein degradation. Kinase and phosphatase inhibitors will be discussed in coming slides. Maintaining RIPA buffer at 4°C can maintain protein-protein interaction, kinase activity and prevent activation of proteases, kinases and phosphatases. Depending on the cell type, additional lysis may be required. This can be carried out by sonicating the cells for short periods. However, it is important to place the cells on ice during sonication steps.

Depending on the protein you are studying, RIPA buffer might be a bit harsh due to the amount of detergent in this lysis buffer, and therefore a different buffer may be required. NP-40 buffer is a less denaturing lysis buffer. However, using NP-40 buffer for immunoprecipitation may result in a higher background, and non-specific protein binding. NP-40 buffer is less likely to inhibit kinase activity and disrupt protein complexes, and also contains similar constituents to RIPA buffer with the exception of Tris HCl, sodium deoxycholate and sodium dodecyl sulphate. Similarly to RIPA buffer, the supplements EDTA, sodium fluoride and phosphatase inhibitors should be added to maintain protein integrity, and post-translational modifications. 

The following cell lysis, pre-clearing your protein lysate, which are control IgG and beads will remove the possibility of non-specific binding in later stages of your IP. Upon lysis of your cells, incubate your lysate with A/G sepharose or agarose beads and control IgG for 1-2 hr at 4°C. Once lysates have been pre-cleared, you can use this extract for your immunoprecipitation experiments.

Next, I will be discussing the maintenance of post-translational modifications. Protein-protein interactions can be dependent on post-translational modifications. Therefore, preservation of protein phosphorylation, ubiquitylation and methylation, the following cell lysis can be important in maintaining protein-protein interaction, and thus producing a successful IP. Ensuring that cell lysis does not result in the removal of post-translational modifications can be imperative, including a range of protease, phosphatase, kinase inhibitors in your lysis buffer can help in the maintenance of post-translational modifications. 

Depending on your protein target of interest, you may be required to add certain phosphatase inhibitors to your IP lysis buffer. Sodium orthovandate, beta-glycerophosphate, okadaic acid and sodium fluoride are commonly used phosphatase inhibitors. Also, it is important to add phosphatase inhibitors fresh to your lysis buffer every time you're about to lyse your cells. This will ensure maintenance of post-translational modifications.

Disruption of the cell membrane can result in the activation of endogenous enzymes such as proteases, which may degrade your protein of interest and affect your immunoprecipitation. Therefore, the addition of protease inhibitors to a cell lysis buffer is highly important to maintain in vivo protein interactions. Due to enzymatic differences of proteases, multiple protease inhibitors may be required in order to prevent protein degradation. The addition of protease inhibitors to your lysis buffer should be sufficient to prevent degradation of your samples. Alternatively, the protease cocktail provides a solution to adding individual protein, in protease inhibitors to your lysis buffer. The protease cocktail can contain numerous protease inhibitors, including PMSF, Aprotinin, Leupeptin and Pepstatin. Although protease inhibitors may be added to your lysis buffer, degradation of your protein sample may still occur. This can be avoided by placing your sample on ice throughout your immunoprecipitation. 

If you're interested in either methylation or ubiquitination of your protein target, inhibition of deubiquitinases or metal transferases with a range of inhibitors, may be required to preserve the in vivo status of your protein. Similarly, to phosphatase inhibition, deubiquitinase or metal transferase inhibitors need to be added freshly to your lysis buffer before lysing your cells. 

Next, I will be discussing IP incubation, washing and buffer optimization. Depending on the disassociation concept of the primary antibody, or the abundance of your target protein, the time it takes for the primary antibody to sequester the target protein can vary. Following pre-clearing of the protein lysate, incubation of the primary antibody with the lysate in a range of 1-14 hr at 4°C, may be required.

Although 90% of your protein may be sequestered by your antibody after 1-2 hr, allowing the reaction to run to completion may be required to ensure total protein sequestration. However, over-incubation of the primary antibody in the protein extract may result in non-specific binding, or protein aggregation. To circumvent this problem you can try to optimize the amount of time it takes to pull down 50% of your target protein without introducing non-specific binding.

Following optimal binding of your target protein with antibody, separating your protein antibody conjugation under protein lysate is the next step. Due to the extra weight added to the sepharose/agarose beads, your protein antibody complex will sediment at the bottom of your marker tube by either gravity, magnetism, or following a short centrifugation. Washing your antibody protein complex is required to remove unspecifically bound proteins, and proteins that may sediment which are sepharose/agarose beads. Similarly to lysis buffer, the wash buffer contains Tris, EDTA, EGTA, sodium chloride, Triton X-100, sodium orthovanadate and a protease inhibitor cocktail.

The primary goal of the IP wash buffer is to maintain desired protein-protein interactions, while removing non-specific protein binding. The addition of a mild detergent such as CHAPs, NP-40 or Triton X-100 may reduce undesirable background. If strong, non-specific interactions persist, increasing sodium chloride concentrations may reduce ionic and electrostatic interactions to disrupt nucleophilic or disulphide bridges, low levels of reducing agents such as, DTT or beta-mercaptoethanol can be added. 

The ideal lysis buffer will leave the proteins in their native conformation, minimizing denaturation of antibody binding sites, while at the same time releasing adequate amounts of protein from the sample for subsequent analysis. However, further optimization of buffers may be required to reduce non-specific background or maintain native function of the proteins. Increasing the salt concentration will reduce non-specific protein-protein interactions. Insufficient lysis of cells may also result in the presence of non-specific background. Increasing non-ionic and ionic detergent concentration will increase cell lysis and nuclear lysis, respectively. However, increasing detergent concentration may also be detrimental to the protein conformation, so further optimization of this step may be required. If you are immunoprecipitating your kinase from a protein lysate, optimization of the divelant cations concentration may be required to maintain full function of your kinase of interest. Increasing the concentration of EDTA in your lysis buffer will help to enable phosphatases. Finally, maintenance of buffer pH will preserve protein function. If you have not used either your lysis or wash buffers for a certain period of time, checking the pH of your buffer may be required.

Following IP wash steps, your proteins and antibody will need to be separated from your A/G sepharose beads. To remove protein complexes from beads, incubate samples at 95°C for 5 min in 2X SDS loading buffer, plus 100 mM DTT. Pellet beads and transfer supernatant to a fresh centrifuge tube. Alternatively, you can incubate your protein complex, I mean, at 65°C for 10-20 min in 2X SDS loading buffer. Pellet beads and transfer supernatant again to a fresh centrifuge tube. This may prevent protein degradation caused by incubation at higher temperatures. 

Separation of your denatured protein from your sepharose beads is an important step in loading your SDS-PAGE gel. Similarly, to your IP washing steps, this can be achieved through the centrifugation of your sample to allow for beads to sediment at the bottom of your tube. Your protein mix will stay suspended in the SDS buffer solution above the beads. Following a short spin, carefully remove the supernatant without disturbing the beads. This can be achieved by running the tip of a Gilson pipette along the side of the centrifuge tube, to prevent disturbing the beads. However, if you carry across beads you can always repeat this process to ensure complete removal of the beads. Once your supernatant has been transferred to a fresh tube, you can now load your SDS-PAGE gel. If beads are loaded into the well of your gel, this may affect the migration of your sample and may affect the appearance of bands following western blot.

Next, I will discuss endogenous versus overexpressed protein immunoprecipitation. Immunoprecipitation of endogenous proteins versus overexpressed or recombinant proteins can yield differences in results. This may be the result from aggregation of recombinant or tag proteins, thus affecting your protein complexes, or overexpression of tag proteins may be above physiological levels. 

As with all techniques, there are both pros and cons. Pros and cons associated with endogenous protein immunoprecipitation, or concentration of endogenous protein may not be sufficient for IP. However, protein-protein interactions will occur at physiological ratios, thus providing a more realistic replication of what happens in vivo. Due to endogenous protein levels, increased amounts of primary antibody may be required to pull down interactions. Thus possibly increase in cost and consistency of the interaction. Finally, your target protein may be the same molecular weight as your heavier light chain band, therefore, masking your interaction by IP. 

Although protein overexpression may overcome endogenous protein issues, there are also some pros and cons. Overexpression of protein may overcome endogenous protein level issues, however, overexpressed protein may be above physiological levels; therefore, may not represent true in vivo interactions. For overexpressed protein, anti-recombinant antibodies may be used to pull down overexpressed proteins. Finally, the addition of a tag to your protein may increase molecular weight of your protein, thus preventing masking by heavy or light chain bands by western blot. Also, anti-target antibodies may also pull down endogenous and tagged proteins, thus increasing your screening capabilities by IP.

Next, I will discuss using bioinformatics to determine interactors. Over the past few years, bioinformaticians have created numerous programs that can predict protein-protein interaction. These programs have been based on data from publications, known interactors, protein sequence, and structural and genomics information. These programs have helped further understanding of biomolecular interactions. However, although not always accurate, some programs may save you time and predict what type of protein-protein interactions you might expect to find. The most popular websites online that help to predict protein-protein interaction are, Eukaryotic Linear Motif Finder, String 9.0, The Biological General Repository for Interaction Datasets, IntAct and the Unified Human Interactome.

Eukaryotic Linear Motif Finder is a computational biology resource for investigating candidate functional sites in eukaryotic proteins. Following the input of your peptide into the ELM tool, your peptide is analyzed for consensus sites of kinases, phosphatases, ubiquitinases, methyltransferases and SUMOylases. Identification of consensus sites will suggest that a possible post-translational modification occurs after sequence, and therefore a particular protein-protein interaction may occur.

String 9.0 is a database of known predicted protein-protein interactions. These interactions include direct and functional associations and they derive from the following: genomic data, high throughput analyses, PubMed, co-expression experiments. Protein interaction with maps may also allow you to further deduce possible interactions that may occur in a pathway.

Next, confirming interactions. X-ray crystallography. Using crystallized recombinant proteins, x-ray crystallography can determine how proteins interact and what domains are involved, thus giving an in vivo representation of biochemical interactions. 

Mass spectrometry. Analysis of protein from immunoprecipitation by mass spectrometry may allow the identification of numerous interactors of the signaling pathway. Furthermore, immunoprecipitation of both endogenous and tagged recombinant proteins can be used to determine protein-protein interaction.

Yeast two-hybrid analysis may allow to screen multiple targets at once. It also has the added benefit of being a eukaryotic system. Yeast two-hybrid may also be used to determine what domains are involved in protein-protein interaction, and therefore allow for further deduction of how proteins interact in a signaling complex. 

A nice technique to screen for protein interactions is yeast two-hybrid, and they're about the pros and cons associated with this. Pros: Yeast two-hybrid allows to screen multiple targets at once. This is an in vivo technique replicating a eukaryotic system. It allows measuring of interaction semi-quantitatively and only cDNA is required of the gene of interest. Cons associated with yeast two-hybrid interactions are post-translational modification in yeast is not identical to human cells. False positives may occur and also maybe some toxicity associated with yeast two-hybrid interactions, therefore, validation of interaction may not always occur.

Following the identification of a protein-protein interaction, further investigation may be required. This can be carried out by mutating suspected interaction domains, consensus binding sequences or structural domains. Deconstructing the protein is also an option to identify interaction readings. However, creation of recombinant proteins may result in proteins losing their natural conformation, and therefore may not represent a true interaction.

To summarize, immunoprecipitation can be used to identify protein-protein interactions. Selection of antibodies is important for pulling down proteins of interest. Optimization of protein and antibody concentration may be required to observe enrichment interaction. Inhibition of post-translational modifications is important to maintain protein-protein interactions. Optimization of wash steps may be required to reduce non-specific binding. Finally, bioinformatics may help predicting protein-protein interactors. Next, I will hand you over to Judith.

JL: Thank you Rachel and Sean for such a detailed seminar. Hello again everyone. I would like to take this opportunity to tell you a bit more about Abcam's IP and Western Blot resources, and products that will help you improve your experiments. Rabbit monoclonals or RabMAbs, are for higher affinity and specificity which provides a high sensitivity, and low background western blot. This makes them ideal affinity reagents for use in pull downs. RabMAbs also offer a diverse epitope recognition of human protein targets and the mouse orthodox, so there is no need to generate a separate surrogate antibody. For further information, please visit

The OptiBlot range includes everything that you need for your western blotting, ranging from protein quantitation kits to ensure equal loading, electrophoresis gels that offer simplified loading, and extended shelf life, to protein ladders and ECL detection kits. For more information, please visit

Immunocapture antibody kits are able to isolate large enzyme complexes in their intact, active state and all of this from relative small amounts of starting material. The antibody in these kits are reversibly cross-linked to protein G-agarose beads, allowing purification of enzymes for subsequent analysis of subunit composition, and/or post-translational modifications.

For superior publication quality western blots, we recommend AbExcel secondary antibodies. All of these 19 secondaries have been extensively tested in the Abcam laboratories, and are available conjugated to alkaline phosphatase or horseradish peroxidase. The dilution range of the AP conjugated products is between 1/5,000 to 1/50,000, allowing you to do more than 1,500 blots with one vial of antibody. For HRP conjugated antibodies, you can do over 600 blots per vial. The average dilution range for these products is between 1/2,000 and 1/20,000. Discover more at

Rachel has already mentioned the light chain-specific and VeriBlot for IP products. These products are ideal for the western blot detection of immunoprecipitated or co-immunoprecipitated proteins. The proteins we normally try to detect with these products are of around 50 kDa, which is the size of the heavy chains, and 25 kDa, the size of the light chains. These products are available for use with mouse, rabbit or goat primaries. For more information, please visit

For quick and easy chromatin immunoprecipitations, we recommend EpiSeeker ChIP kits. Before I explain these products further, I would like to point out that these kits are for cross-link ChIP and can't be used to a native ChIP. Our one-step and plant ChIP kit optimized for mammalian and plant DNA, respectively. These kits do not contain a preselected antibody, and are therefore adaptable to any target of choice. The EpiSeeker range also includes the kits optimized for methylated or acetylated histone modifications. Kits for the immunoprecipitation of methylated DNA are also available. To get an overview of all kits, please go to

To help you with your western blot and IP experiments, we have a variety of free resources, including Introduction to IP, Western Blotting and Fluorescent Western Blotting webinars, Guides for chromatin, UV cross-linking and RNA immunoprecipitation, as well as IP troubleshooting tips. If you are interested in these resources, please drop us an email or click on the links and the PDF that will be mailed to you after the webinar. Also, please feel free to browse our literature and poster libraries at, and download your own copy. Alternatively, you can contact after mailing, mail request at and ask us for a complimentary hard copy.

Abcam’s scientific support team is here to answer any questions you may have. The team members are multilingual and offer support in a range of languages, including French, Spanish, German, Chinese and Japanese. You can contact them in the US, UK, Hong Kong and Japan.

If you would like to meet us in person, Abcam organizes a range of conferences so why not join us on April 24th in London for a one-day symposium on Mitochondria - The Cardiovascular System and Metabolic Syndrome. If you are interested in this meeting, please go to to find out more. Another conference we are running from June 10th to 11th is the New Avenues of Brain Repair meeting at Harvard University. For more information on this event and all our events, please go to

To thank you for attending this webinar, we would like to give you a special 25% discount on all AbExcel and VeriBlot secondary antibodies, EpiSeeker kits, OptiBlot reagents and RabMAbs. All you have to do to take advantage of this offer, is to quote promotion code IPTBIW7 when placing your order. I would like to hand you over to Rachel and Sean now, who are answering the questions you have been submitting during the webinar.

RI: Thank you, Judith. We have received a large number of questions, which is fantastic. Thank you all. For time reasons we will only be able to answer a few of them though. For those of you whose questions haven't been answered, we will contact you by email over the next week or so. So the first question we have is from Gerhardt, and it was: Can we add the protein antibody beads altogether? The answer here is, yes, you can, but it's not best practice. It's best to incubate, bind the antibodies to the beads first, so that you can wash off any Fab fragments, and also any things that are in the buffer of the antibody or the beads. So that's all you add onto your extract is in the buffer you want it to be in.

SG: So our next question we have is from Michael. The question is: Could you please tell me the function of EDTA in your lysis buffers? Well, Michael, there's two reasons why we could add an EDTA to our buffers. Firstly, if would be to inhibit the activity of phosphatases, if this was a problem with your immunoprecipitation and you were seeing dephosphorylation of your protein. Secondly, is to bind calcium and magnesium ions in case these are actually, again, having any effects on your IP of your protein, or any reactants as you're subsequently doing.

RI: Then we've got a question from Kaya who was wondering what the advantage was of using covalently bound kits that binds your antibody without the need for protein A or G beads, or worrying about the isotype. It's a very good question, so these covalently bound kits can be really good, because as the antibodies are bound covalently, when you do the elution the antibodies stick to the beads, and so you won't have any interference in the western blot. Another advantage is that the antibody beads ratio has already been determined for you, so you can just take them out of the fridge and use them.

Then we've got another question from Sarah, which is: What's the advantage to using magnetic beads instead of agarose beads? So there are quite a few advantages, actually, for using magnetic beads. One of them is if you use magnetic beads the magnet will attract the beads to the side of the tube, which means that it's quite easy to take the supernatant off. So if you have an assay where you really have to take all of the supernatant off, magnetic beads are a good choice. Also, you have no centrifugation steps, and as magnetic beads are really tiny you can have very short incubation times, which is sometimes a good thing if you've got proteins that are not very stable.

Then there is another question from Ishmael, which is: Can we use the same antibody for western blot as was used for the IP? Here I would say, yes, you can, but first you have to ensure that it actually works for western blot, because not all antibodies do. Then you need to select the right secondary reagent for this, so we suggest using light chain antibodies for all the proteins that are bigger than 30 kDa, and VeriBlot for IP for anything that is smaller.

SG: So our next question is from Anya, and her question is: For a co-IP, what guarantees the two proteins that are bound to each other, are bound specifically? Now, with IP there's no guarantee, Anya, unfortunately. However, it is a good indication that a biochemical reaction, or interaction might be occurring. However, with IP there may be further validation required, so you can further validate this by, as I mentioned, x-ray crystallography, yeast two-hybrid interactions, possibly FRET. You could also further validate by checking the interaction cell cycle dependent. You could inhibit the pathway using small molecules inhibitors. These can be got from Abcam Biochemicals. Finally, you can try reverse reactions to see if you pull down the opposing protein will the interactions still occur?

Another question that I've had through our Facebook is from Hernando, and the question is: Hernando would like to hear how we'd recommend a higher peptide or protein recovery from an antibody following immunoprecipitation? So, Hernando, there are three common methods either using urea, STS or glycine buffer elution techniques. STS buffer's the harshest and will also elute non-covalently bound antibodies, and antibody fragments along with your protein of interest, a result of nearly total protein recovery. However, if you're planning on carrying out biochemical experiments, which are IP'd protein, we would recommend that you possibly stick with glycine. Alternatively, you could use your real buffer and this may be useful if you plan to carry on mass spectrometry, as your re-elution buffer is compatible with protein digestion. So that's all the questions we have time for. As Rachel mentioned, hopefully we'll be able to answer your questions in the coming days by email. Finally, I will hand you over to Judith.

JL: Yes, and I think this is the time you've all been waiting for, it's the answers to the quiz. About question one: Which Rat IgG isotype does protein A bind to? The correct answer here is answer four: IgG2c. If you would like to review this again or you've got this wrong, we'll send you the slides after the webinar so you can go back to slide 14, which actually lists when to use which type of IgG binding protein. Or, alternatively, you can go to our website to go onto the isotype table. With regards to question two: A protein is an inhibitor of which protease? The correct answer is number one, it's a pancreatic trypsin inhibitor. Now, question three: You have immunoprecipitated superoxide dismutase 2, which is 25 kDa protein. You've done this with a rabbit polyclonal antibody, and want to use the same antibody that you used in IP for western blot detection. Which secondary reagent do you use? You thought a lot about these reagents, because the correct answer here is secondary number four: The anti-rabbit IgG VeriBlot for IP. We recommend this product as this only recognizes the native, non-reduced primary antibody. Again, if you would like to know more please go to

I would like to finish by thanking Rachel and Sean for the seminar, and all of you for attending. Thanks a lot for all of your questions, and I hope you join us in the near future for another Abcam webinar.

Ladies and gentlemen, this concludes today's web seminar. We appreciate your time and attention, and it is now safe to disconnect. Thank you and have a nice day!

Seán Garry
Seán received his degree in genetics from Trinity College Dublin in 2006 and continued into research by doing a PhD in the laboratory of Dr. Margaret McGee at the Conway Institute, University College Dublin. For his thesis, Seán investigated how the pro-apoptotic Bcl-2 family member, Bim, is phosphorylated following prolonged mitotic arrest. After his PhD Seán relocated to the laboratory of Dr. Viji Draviam at the Department of Genetics, Cambridge University, to carry out post-doctoral research into how kinetochore-microtubule attachment is established during mitosis. Seán joined Abcam in 2012.

Rachel Imoberdorf
Rachel studied Biology at the University of Bern, Switzerland. After this, Rachel moved to Geneva, Switzerland where she did her PhD in Molecular Biology in the laboratory of Prof. M. Strubin. During her thesis Rachel was interested in understanding the effect of global histone acetylation on transcriptional regulation in the yeast S. cerevisiae. She also worked on the development of a system to measure transcription factor turnover on specific regions in the genome. Rachel joined Abcam in 2005 as Senior Scientist and manages a team of 8 people who establish and optimize techniques and processes in the Abcam laboratory, including Immunoprecipitation (IP) and Chromatin Immunoprecipitation (ChIP).

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