Finding The Right Antibody

Before you begin

Remember, it’s important to take your time

Not all antibodies will be suitable for your experiments so taking a bit of time choosing an antibody can stop you wasting anything from a few days to several months at the bench. This is especially true with new researchers - you don’t want to end up blaming yourself for a failed experiment, when in reality it is the antibodies fault! Estimates vary, but it is suggested that up to half of commercial antibodies may not be fit for purpose [ 1 , 2 ]. With such a high proportion of antibodies bought not working and with the massive amount of choice out there, this is not a process to be rushed.

Selecting an antibody

Starting with an antibody used by your peers

Taking a look at antibody citations, records of how antibodies have been used in previous scientific papers, is a sensible place to start in your search. This is a great way to get a view of which antibodies are available and being used by your peers and complements valuable supplier provided information.

Choosing well cited antibodies has many advantages. Firstly, you will reduce the risk of choosing an antibody that gives no results as you can easily see where and how the antibody has worked in the past. Publications provide really valuable third party independent data on which to evaluate the antibodies performance, although it’s important to ensure data quality and detail when doing so. It may be worth looking at cited images using the antibody. You can evaluate the approaches used, giving you the confidence you need in the product.

An antibody search engine is one such way to do all of this in one centralised resource. At CiteAb, our search engine ranks by citations, and includes experimental details and images.

However, do proceed with caution. We must stress that the most cited antibody might not be the best for your specific experiment. It is a useful starting point, but there are many other important factors to consider such as validations, application, species and more, which we discuss below.

Is your antibody validated?

It is important that the antibody you select is well validated, see ‘After Purchasing’ for further details of different types of validation. Validation will give you confidence that the antibody you use is specific, sensitive and able to give you reproducible results. Reviewing the published literature from your peers, or looking into what validation suppliers have carried out, can show you how the antibody has been validated in previous work. You may still need to plan additional validation in your lab after purchasing.

A common issue in the search for research antibodies is their ability to bind to alternative targets to what you are testing. Some may even bind to different targets with a greater affinity.

Is your antibody suitable for your application and species?

It is very important to consider if the antibody will work for the experiments you have planned. Just because an antibody has been successfully used for western blotting does not mean it will work for flow cytometry, for instance.

The majority of antibodies are raised against human proteins so if you are not working with human samples be careful to check if the antibody will cross react with the species you are using.

Check the antibody host species

It’s very easy to use the wrong secondary antibody and a blank western blot is so painful, so make sure you check which species your primary antibody is raised in. This is especially important if you are doing double staining and need two different species of secondaries. If using goat antibodies it is also worth checking if your secondary antibodies are raised in a goat and may cross react.

Monoclonal vs polyclonal vs recombinant

When choosing between these our advice is you should go with the most validated antibody that works in your application and species. However, if you are lucky enough to have several well validated antibodies to choose between, then monoclonal antibodies may suffer from less batch to batch variability than polyclonals (although you can’t assume no variability). Polyclonals from large animals like goats may also have less batch to batch variability.

Recombinants offer the advantage of high reproducibility. Through expression of a defined antibody-coding sequence, genetic drift seen over time with hybridomas is avoided. The known sequence of a recombinant antibody can be re-synthesised and re-expressed, also minimising issues with batch-batch variability.

New antibodies

It is also worth considering new antibodies. These will not be as highly cited, but may offer potential benefits not yet shown in the literature. Comparing these to the most cited antibody through tests in your lab could illuminate which is the best antibody for your experiment.

Practicalities of purchasing

Several companies may sell exactly the same antibody

Many manufacturers licence their antibody to multiple different companies, so do not buy five antibodies to test without making sure they are actually different. You can do this by asking where the antigen is found within the protein or checking they are raised in different species. With common monoclonals that are supplied by many companies you might also consider shopping around for the best supplier of a clone.

Price considerations

Small price savings on an antibody may not be worth it in the long run. Wasted time is expensive in the lab, so getting the antibody that is most likely to work is priority number one. However, this is not to say a less expensive antibody won’t work as well.

Do you have a preferred supplier?

If you’ve found a preferred supplier that works for you and provides high quality antibodies consistently then it may be worth looking to see if they have an antibody for your target. You could compare this to the most cited antibody and independently test the performance of both in your lab. It is worth bearing in mind that some organisations will not allow you to purchase from certain suppliers - this could be due to animal rights issues or other ethical concerns.

After purchasing

Validate the antibody in your lab

After checking what validation has already been done on your antibody from the literature, it is important to also test the antibody yourself. There are many ways to validate your antibody, and many resources out there to help you do so. You could consider: genetic strategies, orthogonal strategies, independent antibody strategies, expression of tagged proteins and immunocapture followed by mass spectrometry 3 .

The level of validation you undertake may depend on how well validated the antibody already is, and how critical the antibody is to your experiment. Is the antibody just one of a panel used to identify a process? Perhaps you don’t need extensive validation. Does your seminal finding rest on its specificity? It will be vital to ensure the antibody is well validated.

Test multiple antibodies

It may be worth buying multiple antibodies that you think could be suitable for your experiments, which you can then test independently in your lab to decide which is the most suitable. As above, ensure you validate each antibody you test.

If the antibody does not work ask an expert

Do the obvious checks to make sure you did not forget a key reagent and then ask for advice - don’t just keep doing the experiment again. Find somebody in your department or email an author who has used the antibody. Also contact the company for help, see below.

If the antibody does not work contact the company

Most suppliers ultimately want to help you in your research. You should feel free to contact the company for advice and even a refund. The suppliers should be helpful, the majority we work with are, and they have a great deal of expertise on their products. Many pride themselves on their customer service - if you have found them to be particularly helpful you can remember this for next time you purchase an antibody.

Helping others find the right antibody

Help others in the research community by citing antibodies you use properly! Once you get your great data and write it up, it is important these antibodies can be identified correctly so others can reproduce your findings - we have a guide here to help you do that.

If you have any questions about finding antibodies to use in your work, or you think we might have missed something, please do drop us a message using the contact form or by emailing us at hello@citeab.com

Additional resources

1. The European antibody network's practical guide to finding and validating suitable antibodies for research EPMC® ID: PMC4966524
2. The Antibody Society: Antibody Validation Webinar series

References

1. Weller, M.G. (2016). Quality Issues of Research Antibodies. Analytical Chemistry Insights, 11, p.ACI.S31614.
2. Berglund, L., Björling, E., Oksvold, P., Fagerberg, L., Asplund, A., Al-Khalili Szigyarto, C., Persson, A., Ottosson, J., Wernérus, H., Nilsson, P., Lundberg, E., Sivertsson, Å., Navani, S., Wester, K., Kampf, C., Hober, S., Pontén, F. and Uhlén, M. (2008). A Genecentric Human Protein Atlas for Expression Profiles Based on Antibodies. Molecular & Cellular Proteomics, 7(10), pp.2019-2027.
3. Uhlen, M., Bandrowski, A., Carr, S., Edwards, A., Ellenberg, J., Lundberg, E., Rimm, D.L., Rodriguez, H., Hiltke, T., Snyder, M. and Yamamoto, T. (2016). A proposal for validation of antibodies. Nature Methods, 13(10), pp.823-827.