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Welcome to our training series on how to properly choose and use antibodies. Here we’ll guide you from choosing which antibodies best suit your needs, how to handle and store antibodies through to validation, and how to troubleshoot when things go wrong.
Now that you know which antibody you need for the right application, and have probably already aliquotted and safely stored it, you can think about how to validate that antibody. And from there, you’re probably going to want to take steps to choose your controls and optimize your setup to get the best possible results.
Antibodies are intrinsically variable reagents and so it’s important you take the time to ensure that the antibody performs as expected in your experimental setup. A good supplier will have already tested the antibody but it’s impossible to account for the huge number of different protocols and reagents that it may get used with once in the hands of the researcher. Your own validation steps are therefore so essential because they’re specific to your setup.
Below are just a few of the most commonly used methods for validation.
Protein-encoding gene eliminated with genetic tools (eg CRISPR)
Protein detected and quantified in a sample via initial separation by size and then blotting onto a membrane to be detected by an antibody
IHC and ICC
Protein in tissues (IHC) or cells (ICC) detected via specific antibodies and reporter molecules
Exactly which controls you need for your experiment will depend on the assay you have planned. However, whichever experiments you choose to run, you should always include positive and negative control where possible. We know that can seem tedious at times, but it really is the only way to give validity to your results. So, get in the habit of using proper controls right from the start. You’ll thank us in the long run.
Tissue and cell controls
We know that it can be hard to find relevant cell lines or tissues to use as controls and it might mean you need to do a bit of a literature review. But before you either order your knockout cell lines or trawl through the publications, here are some databases that have useful expression data and serve as a great starting point:
Watch the below video on how to search these databases:
Recommended controls for western blot
Looking to optimize your western blot experiment? Find out about recommended controls for western blot.
Controls for IHC
It is essential to run controls in IHC staining experiments to confirm that the observed staining pattern is true, accurate and reliable.
Controls for flow cytometry
Improve your flow cytometry results by using the appropriate controls.
Application, check. Antibody, check. Validation, check. Controls, check. Everything’s in place. The last step now is to optimize your antibody-based assay. The main reason for this is to make sure you visualize only specific staining and reduce any non-specific background signal. Things to consider here include incubation times, blocking conditions, choice of buffer, and antibody dilution.
Your antibody should come with an optimal dilution on the datasheet, but this might not be optimal for your setup. For this reason, you should establish which concentration gives the best signal. The most practical way to do this is by using a series of dilutions in a titration experiment. For example, if a product datasheet suggests using a 1:200 dilution, then we suggest making dilutions of 1:50, 1:100, 1:200, 1:400, and 1:500.
If you have an unpurified antibody and don’t know a suitable dilution to start with, you can use the table below to get going.
Western blot/dot blot
While heading down the optimization route, it’s worth taking the time to optimize how long you incubate your antibody, how you decide to block (BSA, casein, serum, commercial blocking reagents, etc), and the types of buffers you’re using. These steps will help you to generate accurate data.
A little time invested at the beginning could potentially save you weeks further down the line.
These protocols provide you with the steps to follow for sample preparation, incubation with your antibodies, and visualizing your data. For best results, we have also provided optimized protocols, including recommended dilutions on the datasheet of our antibodies.
Get your protocol guides below
Next week, in the concluding part our series, we’ll give you answers to the common problems encountered in immunoassays like western blot, IHC, and flow cytometry.
You’ll learn how to solve the following problems:
We’ll also give you several detailed troubleshooting guides to other applications to help keep your research on track.
In case you missed or want to revisit the earlier parts, you can find them here: