Five Tips for a successful antibody experiment

Written by Raquel Pinho | Sep 5, 2019 6:55:17 PM

Ain’t nobody got time for bad antibodies! 

Since the 20th century, antibodies have become pivotal to basic research, health diagnostics, and drug development. However, they are also viewed as the culprits of most major frustrations in the lab. Despite being routinely used in a wide variety of laboratory techniques, finding a high performing antibody is a challenging task. Every year, energy, time and money go to waste due to poorly performing antibodies and unreproducible assay results, with loss estimates exceeding US$800 million - as reported by Andrew Bradbury & Andreas Plückthun

But why is it so hard to find a good antibody?

On one hand, the business of research antibodies grew impressively in the last decade. Scientists can now choose from more than 300 suppliers worldwide, with more than two million research antibodies on offer. On the other hand, this escalating number does not go hand in hand with clearer quality standards. 

Never assume an antibody is fit for purpose just because it is commercially available.

There are a variety of issues that may cause an antibody to fail, including high variability between different batches produced in animals and a lack of clear and enforced validation standards. Antibody experiments can rapidly become maddening roller coasters, but planning your experiment scrupulously and knowing how to research and compare different antibodies will greatly increase your success rate. 

Life is too short to buy useless antibodies!

Want to find the best antibody for your next experiment? Check out our TOP FIVE TIPS to guarantee you end up with reliable data. 



Top Five Tips for a successful antibody experiment

Tip #1. List your antibody requirements

Tip #2. Search for commercially available antibodies

Tip #3. Review relevant literature and protocols

Tip #4. Check published validation results

Tip #5. Run proper control and validation experiments


Tip #1. List your antibody requirements 

Define your basic primary antibody requirements 

Planning is the most important phase of an experiment. Start by understanding and defining the basic specifications your antibody should have. 

Antigen & epitope

Find out the official name of your protein of interest. Antibody suppliers may use different synonyms and alternative names, so make sure you check public databases, such as NCBI and GeneCards, to find all aliases. Choosing a good antigen and a specific epitope (i.e. a region in the antigen with low similarity to other protein sequences) is critical. 


When detecting a truncated version of your protein you will not be able to use an antibody that binds the missing amino acids. Also be aware that protein conformational changes (e.g. when using tagged proteins) may mask some epitopes and prevent binding to your antibody. 


Reactivity, specificity & cross-reactivity

The species of your protein of interest defines the antibody’s reactivity. To determine specificity/cross-reactivity, check protein homology in BLAST. This tool enables you to identify unique epitopes of an antigen and regions where antibodies may cross-react with other proteins with a similar sequence. If the species of your target protein is different from your samples (e.g. expressing a human protein in a mouse cell line), make sure to evaluate any potential cross-reactivity between species. 

Application and type of samples

It is vital that you decide whether you need to use a qualitative (e.g. immunohistochemistry [IHC]), or a quantitative technique (e.g. western blot), or even a combination of the two. When choosing, take into consideration your sample type (e.g. not all techniques work with paraffin-embedded samples).

Conjugate & host

It is possible to study your protein of interest via either direct or indirect methods. When using an indirect method make sure to choose a host species for your primary antibody that is compatible with the secondary antibodies in your lab. When choosing what type of conjugate to use, take into account the selected experimental technique and the detection equipment available.

Sensitivity & clonality

The expression level and structural variability of your protein make decisions on antibody clonality and sensitivity critical to your experiment. For example, when detecting low abundance proteins with high structural variability consider using polyclonal antibodies, as they are more sensitive than monoclonal antibodies, despite being less specific. 

Are some of these terms new to you? Check out our knowledge box!


Calculate how much antibody you will need 

Define the amount of antibody you will need to purchase to run the full experiment. This will ensure you use antibodies from one single lot number, therefore avoiding batch-to-batch variability. 

Choose the right controls 

Including the right controls in your experimental design is a key determinant for a successful experiment. Define controls which are:

  • Relevant for your experimental setup (e.g. using samples expressing [positive control] or not expressing [negative control] your protein of interest).
  • Specific to your technique (e.g. for IHC include a positive, negative and secondary antibody-only control to distinguish between true labeling and background, for western blots use loading controls, and for ELISAs perform standard curves). 


Tip #2. Search for commercially available antibodies

Use life sciences marketplaces & search engines

With the increasing number of antibody suppliers, finding the best and cheapest antibody matching your requirements can be a daunting quest if you use traditional search techniques (i.e., searching through the literature and visiting each antibody supplier’s website). A quicker and more efficient way to find antibodies is using search engines & life science marketplaces. Some of the most robust platforms include ZAGENO (marketplace), antibodies-online (marketplace) and Biocompare (search engine). These platforms allow you to browse and search for all available antibodies for your protein of interest, from different manufacturers, with just a few clicks. Taking full advantage of the digital era can help you save valuable time and may introduce you to new suppliers. 


Filter by antibody specifications

Marketplaces allow filtering of search results by antibody specifications (i.e., you can tailor your search according to your specific antibody requirements, such as application, reactivity, clonality, host, conjugate, and others). After selecting these filters, all commercially available antibodies matching your preferences will be displayed. 


Compare available antibodies

An important step is to find the advantages and disadvantages of each available antibody. The easiest way is, again, to use marketplaces. There are two comparisons to keep in mind: 

  • Compare antibodies from different manufacturers. You can select the antibodies matching your requirements and compare them side by side. As the comparison view will display the main product specifications, this is a fast way to spot important differences between two antibodies (e.g. the recommended dilution, for IHC, could be 1:100 for one antibody and 1:500 for the other antibody). 
  • Compare antibody prices when purchasing from different suppliers. You can also use marketplaces to quickly find the cheapest available price for your specific antibody, as prices from different suppliers are displayed on one product page. 

At this point, you may have found several antibodies with similar specifications. How can you figure out which one is more likely to work in your experimental setup? Tips #3 and #4 are the key!


Tip #3. Review relevant literature and protocols

Gather relevant literature. Filter by experimental context

Public databases, such as PubMed and GoogleScholar, are a great source of scientific literature but not ideal for antibody searches. Life Sciences marketplaces & antibody search engines, on the other hand, were designed to help narrow your antibody literature search. For each antibody, you can immediately check if it was cited in a publication. To increase the chances of success, focus your literature review on studies using similar experimental contexts as yours (ie. same application, sample, sample species). 

BenchSci and CiteAb (two search engines) allow you to filter the listed papers by specific experimental contexts (e.g. application or species), so you do not have to go through each and every paper.

Carefully inspect antibody data 

A common pitfall is to assume that if an antibody was used in a publication it is of high quality. Often, data provided in scientific journals is incomplete. Meticulously scrutinize antibody data; always check if appropriate positive and negative controls were included, as well as a comprehensive protocol. Be wary of cropped blots and antibodies showing inconsistent results across the literature (e.g. different expression patterns between studies using the same sample type). 

Get in touch with the authors! Not only can they give you more details regarding antibody validation and performance, but they can also provide valuable troubleshooting information.



Check product data sheets and manufacturer's instructions 

Antibody data sheets can be found on the antibody’s page on a marketplace, or on the manufacturer’s website, and generally, include detailed information on the antibody as well as relevant protocols. If the manufacturer recommends using the antibody for an application but does not provide a detailed protocol, get in touch with them. Several manufacturers and marketplaces offer live chat services, allowing instant and easy access to support. Request tips on how to use your antibody for your specific experiment and ask if they can help you with troubleshooting in case the antibody does not work.


Understand the protocol and review all required material 

Once you get a protocol - from the literature or the manufacturer’s data sheet -, make sure you understand what the critical steps are and whether you have all the necessary protocol details and materials. Like most things in science, details are key. Even when you buy a great antibody, you can get completely different results by introducing small protocol variations (e.g. changes in the water’s pH, using different membranes and blocking solutions, loading different amounts of protein or different samples, choosing different settings or using a different detection machine). 

Documenting any deviations from the original protocol will be very handy if your antibody does not perform as expected and you need to troubleshoot. 



Tip #4. Check published validation results

Look for validation data from different sources

You dramatically increase your success rate when you select an antibody that has been validated for your specific application and samples. Unfortunately, a high percentage of available antibodies have not been properly validated. Some initiatives are trying to reverse this trend and inspire both manufacturers and scientists to share information on antibody validation. This is the case for antibodypedia; an open-access gene-centric antibody database that compiles extensive validation data, the Independent Validation Initiative; from antibodies-online, that refunds the antibody purchase price in full if the scientist shares their data, and ZAVI; ZAGENO’s initiative, that offers open access to structured validation data generated by researchers and antibody suppliers. When validation information from the manufacturer is not available, visit these initiatives to access the results obtained in specific experimental contexts.


Review available validation data 

In order to be useful, validation data should include a detailed validation protocol, relevant controls, uncropped images and, ideally, show results in multiple sample types. Get familiarized with the challenges and possibilities for antibody validation. Some good literature on this topic includes this paper from Michael G Weller and the Abcam validation guide. Briefly, antibodies should be validated in an application-specific manner. The main pillars of antibody validation include, but are not limited to: 

  • Genetic validation, by knocking out or knocking down your protein of interest 
  • Orthogonal validation, where the target protein is studied using an antibody-free and an antibody-based technique 
  • Independent validation, by using different antibodies to examine your protein 
  • Biological validation, where the antibody specificity is demonstrated by modulating the expression levels of the protein 

For your experiment, select antibodies that have been validated for your species and application. When validation data is not available for your application, it’s worth getting in contact with the manufacturer to understand whether the antibody failed the validation or if it was simply not tested for your application. 


Sometimes, a reference is provided instead of validation data. In these cases, make sure that you check if the antibody was actually validated for your specific technique and samples in the original study.



Tip #5. Run proper control and validation experiments

Do a test run and validate each antibody

Once you choose an antibody, there are a couple of additional tips that can help you perform a successful antibody-based assay. Number one tip: always start by testing your antibody for your specific application, looking at specificity, sensitivity, and reproducibility, especially when no antibody validation data is available. Resist the temptation of skipping this time-consuming task! Keep in mind:

  • Available validation data may have been generated with a different antibody lot number Antibodies may perform differently in your hands/lab You might be using a slightly different protocol than the one described 
  • Test the antibody and get comfortable with the protocol before performing your experiment with important samples. 

Always use controls 

An experiment without controls is like a boat on dry land: useless. To interpret your results correctly, you need controls. Controls are also useful for troubleshooting when you don’t get the expected results. For example, if no signal is detected for your samples, but your positive control gives a strong band, you can almost certainly exclude any potential errors caused during antibody incubation. More importantly, with the right controls, you can be confident that your results are specific and reliable. 


Store and handle your antibody correctly 

This means not only following the protocol correctly and documenting any departure from the original method, but also making sure to store your antibody properly. Check whether you can use preservatives (e.g. azide) or if your antibody can be defrosted multiple times. Some antibodies should be aliquoted and stored at -20°C, others at 4°C, whereas some are light sensitive and require storage in the dark. 


Make no mistake: working with antibodies is tricky!

Even when you follow all procedures correctly, you may still end up struggling with empty blots. This may not necessarily be due to your antibody performance. For example, it may be that your sample is degraded or your transference settings are not appropriate for your protein, especially when dealing with very large or very small proteins. The good news is that there are dozens of great troubleshooting guides for most applications. Some of our favorites are from Bio-Rad, Abcam, and StressMarq Biosciences.

Hopefully, our tips and checklist will help you find the best antibody for your experiment quickly and easily, while also achieving reliable results. We highly encourage you to take full advantage of all the digital tools mentioned here and to always ask for help! - whether from suppliers, specialists or your team members. To learn how Zageno can better support you, book a live demo here

Best of luck with your experiment!