How do you pick which antibody you should use in your assay? If you’re starting a new assay and need an antibody for the job, or if your assay isn’t working and you suspect that your antibody could be at fault, then selecting a new antibody from the plethora available could be high up on your to-do list.
But how to do this?
Antibody quality for research antibodies is not regulated in any way and so the antibody market is flooded with antibodies that do not recognize their supposed target at all, some cross-react recognizing multiple proteins while others work, but only under very specific conditions. When selecting an antibody there are a few things to consider; your PI will tell you the right price is imperative, but repeating an experiment and buying a second or even third antibody because you got it wrong the first time around is going to cost you much much more than getting it right from the get go.
Here’s where a bit of antibody know-how comes in. Rather than sending you to root through your undergrad immunology class notes, this list offers you a quick step-by-step guide to help get your assay off to a bright start.
Primary antibody target acquired
The first place to start is your target. Ask yourself the following questions:
What protein are you interested in identifying?
Does it have to be the whole protein, or perhaps a specific section such as the C-terminus?
Do you want to detect a modified version of the protein such as a phosphorylated or ubiquitinated form?
Then, make sure your antibody specifically recognizes your exact target or your assay results will be giving you answers to a different question than you asked.
For some targets you might find a plethora of available antibodies while for others you might be restricted to just one or two. If you are really struggling finding an antibody for your target, you might have to consider a custom antibody to achieve your results.
Primary antibody species specificity
Next check that the antibody to your target is reactive with your species. If you are working with a human cell line, then the antibody must recognize the human protein. Likewise, if you are working with a mouse model your antibody must be murine reactive. Get this wrong and you won’t be able to pick out your protein.
Polyclonal or monoclonal primary antibody?
Now you can start to whittle down the options based on your experiment. If you want to identify a protein which isn’t expressed at high levels, or need an antibody for western blotting, IHC or IP, then a polyclonal might give you more success, as they can bind to multiple epitopes on the target protein. If your target is a conformation specific epitope or you are planning to use multiple antibodies to identify different targets within the same assay, then a monoclonal is your best bet, offering increased specificity and lower background.
Is the primary antibody tailor-made?
You think you’ve found a few good options- now you need to decide which is for your experiments. Check the antibody datasheet to see if it has been approved for use in your intended application- an antibody that works in western blotting might not work in flow cytometry or IHC.
Carefully check how the antibody was validated though. Did the company use a recombinant protein on the western? Antibodies often react differently to a cell lysate and might not give the same clean result.
You ideally need your technique covered at this point, but if that’s not possible in your antibody choices, opt for an antibody that works under similar conditions- an antibody that works in native IPs might work in flow as they both preserve the native protein structure.
Has the primary antibody been road-tested?
Quality citations are important. Check the references on the datasheet. While the antibody manufacturer might include any and all references that mention the use of their antibody in the literature, these citations aren’t always good. When you check the references you could see images that don’t look like anything you would want to receive from your experiments, so citations alone aren’t good enough, quality citations are where it’s at.
To go hand-in-hand with this, you should check PubMed for references to an antibody, to see if people have used it as you plan to, potentially giving you great tips on working dilutions, etc. There are now additional databases of antibody reviews, like pAbmAbs, Antibodypedia and CiteAab, which compare good antibodies and where users offer reviews for specific applications that might help you to decide. Do try and make sure though that you obtain the same lot number as used in the citation and reviews. Batch-to-batch variation in antibodies can be huge and so getting hold of a different lot number can mean a completely different antibody.
Beg, steal and borrow a primary antibody
Finally think about the experiments you have planned. If you only want to run a one-off western then do you need to buy a whopping great aliquot of antibody? Could you get away with a cheaper trial size aliquot? Ask the antibody companies for discounts, guarantees that if the antibody fails they’ll give you your money back or an alternative to try, or even a free few microliters of antibody. You can get away with all sorts when you try.
After all that hard work selecting the right antibody you should still validate it in your own system. Antibody companies do not guarantee that an antibody will work for every tissue type and set of conditions. Unless you are fortunate enough to have someone else prove specificity in the same tissue from the same species under the same experimental conditions, you will have to do this yourself!
So you want to work with mouse B cells? Primary murine B cells are a difficult, yet fascinating system to work with and can help deepen your understanding of an immunological system. You can study many things with primary B cells, including: immune activation antibody production cell-cell interactions between immune cells and immune phenotype These […]
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