Scientific Support Says: What’s in Your Vial Matters

Nicholas McCurtin is a chemical biologist with a PhD in Chemistry from Tufts University and an expert in PTMs like phosphorylation and ubiquitination. Having run countless western blots and immunoprecipitation experiments, Nicholas brings first-hand technical expertise directly to the bench. Read below for practical, researcher-to-researcher advice in this edition of the Scientific Support Says column.

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Have you ever thought about what’s inside of your antibody vial? I’m sure you have looked inside of the vial and pondered how many microliters you have left, but I am wondering if you’ve ever thought carefully about the formulation of your antibody.

When I was at the bench, this isn’t something I ever really thought carefully about. That was fine, since I only really used antibodies for western blotting. Had I been doing work with live cells or animals? I would have been done for.

At HUABIO, we get it, you are probably too busy pipetting to really relish in the different types of antibody formulations. For the most controlled experiment, though, it becomes important to keep track of what’s inside your vial!

The typical formulation of an antibody contains several ingredients. Of course, you will have your antibody in the vial. That is a non-negotiable. Your antibody is dissolved in a buffer such as PBS, TBS, or HEPES. In some cases, antibodies will be lyophilized and diluted by the end user!

Like most proteins, however, antibodies are quite sticky when in a plastic environment, so many antibodies are formulated with what is called a carrier protein to coat the plastic of your tube so that less antibody will be lost to plastic-binding. The usual suspects for carrier proteins are albumins, like BSA, HSA, and OVA, but at times gelatin will be used as this sacrificial protein. 

Your antibody also is usually packaged with a cryoprotectant in solution, typically via a sugar like glycerol that will allow you to freeze-thaw your antibody with less damage done by the formation of ice crystals.

Finally, your antibody is often stabilized by an antimicrobial agent. Historically, sodium azide was the primary bacteriostatic included in antibody formulation, but many companies are moving towards the use of alternative, greener reagents like ProClin 950.

Were you surprised that so much was going on in these little tubes? I know I was. Again, I was just a blotter, so it was less about what was inside of the antibody and more about how the antibody looked when I made these decisions as a bench scientist. If you are at the bench right now, you might want to consider these components a bit more carefully when purchasing an antibody. 

What I mean by this is you have the power to shop your formulation. When you look for antibodies, you can scope out whether your product is carrier-free, BSA-free, azide-free, and so forth. Basically, you could either get an antibody with all of these additives – which are not bad, just incompatible with certain assays – or you can choose which you want to leave out. 

For example, if you are trying to conjugate something to your antibody, you would probably want to leave out proteins that are not your antibody. With sacrificial BSA or other carrier proteins, your conjugation yield will decrease thanks to competition with the carrier protein. In this case, you would want to seek BSA-free antibodies to perform your conjugation experiments. It will still have azide and glycerol, but those won’t necessarily compete with your antibody as a starting material. 

So when would you want to leave the other components out? 

Sodium azide is toxic, so if you are performing live cell assays like flow cytometry or immunofluorescent microscopy experiments, you would probably want to leave this component out so as to not cause cell death that interferes with your measurements. Be sure to double-check when you purchase an azide-free antibody, alternative bacteriostatic agents can also be cytotoxic!

Carrier-free antibodies are typically free of both carrier proteins and bacteriostatic agents like sodium azide. For more sensitive conjugation and live-cell experiments, this grade of antibody will typically be sufficient. If you really are really concerned about additives like glycerol, you could even consider a PBS-only antibody. PBS-only antibodies are suitable for IP-MS, SPR, and in vivo micro-injections, all of which would be complicated by typical antibody formulation.

Which one should you choose?

That is highly dependent on your application of interest. You should consider your experimental setup and the biological parameters of your system. By considering both chemical and biological impacts of the non-antibody components within your antibody tube, you will be better suited to get the most out of your time, samples, and funding. Not sure which formulation is right for you? Contact us at [email protected] and we will evaluate and talk you through it!