When it comes to primary antibody selection, these are the things you need to think about to get the most from your experiments.
It’s no secret that antibodies against membrane proteins, like G-protein-coupled receptors (GPCRs) and ion channels, are attractive for both basic research and drug discovery. But finding an antibody that works consistently can be a bit of a challenge. Usually, this is down to the difficulties in antibody generation, i.e., antigen presentation (or lack thereof!) and subsequent antibody selection.
Luckily for you, we’re membrane protein specialists. In an upcoming blog we’re going to be talking about how we get hold of those pesky membrane antigens and generate reliable antibodies against them, but for now, rest comfortably in the knowledge that this step has been taken care of by professionals.
With us handling the antibody generation bit, here are few more points you need to consider before you choose your primary antibody.
What assay are you running?
This should be nice and easy. Are you running immunohistochemistry? A western blot? Flow cytometry? Maybe you’re running several assays. Whatever it is, make sure you know this, and you check that the antibody has been validated in that assay.
Most vendors will validate an antibody in multiple applications, which means they’ve tested that antibody and are confident that it works when used in that situation. Because different applications across different assays expose your antibody to potentially quite different conditions, you need to know that it has been tested beforehand.
If you look at our datasheets, for example, you can see we’ve validated our Anti-SCN1A (NaV1.1) Antibody (#ASC-001) in ICC, IF, IHC, and WB. We also have good reason to believe it might work in IFC.
If the antibody you’re interested in hasn’t been validated in the application you’re running, there’s a chance it simply won’t produce the results you’re looking for. In which case you might want to keep searching!
What species are you working with?
Since we’re looking at that part of the datasheet, let’s talk about species reactivity. This refers to the species the antibody will react with, i.e., animals in which the vendor knows the antibody will bind the intended target. Just like with the application, the vendor has done a lot of the validation work for you and so you can be confident that if you’re also working on one of those listed species, the antibody will work for you.
However, as we all know, science isn’t always that straightforward. Plenty of us work with non-model organisms beyond the realm of lab rats and mice. A substantial number of researchers work with insects, crustaceans, or amphibians. What about in those cases?
Well, in a lot of instances, major proteins are conserved across species. An antibody should react with the same protein in a species not on the reactivity list if there’s enough homology between sequences.
Unlike some vendors, we can help you out with this because we happily give you the immunogen amino acid sequence. In fact, it’s right there on the datasheet. Other vendors like to keep this to themselves and say it’s proprietary, but we think it’s more helpful for you to have all the available data. This way you can go and BLAST the sequence homology for yourself and decide if you think it’s work trying an antibody in a non-validated species.
Just a quick note on species reactivity and “Host”. Host is the species of animal the antibody was generated, or ‘raised’, in. The reactivity is the animals in which the antibody binding has been tested in. You want to try to avoid selecting a host species that matches the species you’re working with as it could bind to a lot of endogenous proteins that you don’t want it to.
Where is your target protein?
Is it sloshing around in the cytosol or is it sticking out of the membrane? This is important to consider, especially if your protein is a transmembrane protein with some regions inside and other regions outside the cell. For example, if you’re running flow cytometry and specifically looking at a protein region you know is expressed on the surface of your live cells, then you should select an antibody that binds that specific extracellular region of the protein. If your antibody was raised against the intracellular part of that protein, your flow cytometry isn’t going to deliver the results you expected, despite the antibody working as well as it could!
You can easily see which of our antibodies bind to extracellular regions of a protein. And, of course, the immunogen’s listed as well.
Polyclonal or monoclonal?
For some reason, polyclonal antibodies have gotten a bit of bad press. Undeservedly, in our opinion. Polyclonals and monoclonals each have their strengths and weaknesses, and it’s important to pick the right tool for the right job. It’s not as simple as one is good while another is bad.
Polyclonal antibodies come from a heterogeneous population and so they recognize more than one epitope on the target protein and so offer a degree of amplification – useful for detecting low-level expression. Because they can recognize multiple epitopes, they’re less fussy than monoclonals, and so can deal with changes in protein conformation that might happen during things like fixation. Polyclonals are also just a little tougher and tolerate a bigger range of pH and temperature conditions than monoclonals. When it comes to specificity, it’s worth remembering the polyclonals we generate are chosen using just a very short sequence of about 15 amino acids, compared the 50 or so used by some other vendors. This means our polyclonal have a significant advantage when it comes to specificity.
Monoclonals on the other hand are homogenous populations that come from a single B-cell clone. This means that all the monoclonal antibodies bind one specific epitope, making them great for batch-to-batch consistency. That gives them less off-target binding than polyclonals and so also generates less background. Just being a monoclonal doesn’t always assure specificity – that will also depend the type of selection that clone underwent. However, that specificity means monoclonal antibodies are less likely than polyclonal antibodies to bind the same protein across diverse species. If polyclonals are tough, then monoclonals are more ‘delicate’ and thus much more vulnerable to epitope loss that can occur in sample treatments or preparation.
So, consider what you need from your antibody and select a polyclonal or a monoclonal based on that, rather than just flimsy bad press.
Primary + secondary or conjugated primary?
Do you need to use a primary and a secondary antibody or would you be better off with a primary antibody already conjugated to a reporter? These are referred to as indirect and direct methods, respectively.
With indirect methods, the primary antibody binds your protein of interest and then you use a secondary antibody, conjugated to a reporter, to bind the primary antibody. The reporter is conjugated to the secondary antibody. There are more steps with the indirect method, but you benefit from signal amplification as multiple secondary antibodies can bind the primary antibody. On the other hand, the indirect methods can also produce more background than direct methods in some situations.
Direct methods use a primary antibody conjugated directly to a reporter, like an ATTO fluorophore for example. Since you don’t need a secondary antibody for the direct method, it’s quicker, cheaper, and can reduce non-specific binding. However, you may get a weaker signal compared with the indirect approach, especially if the protein of interest is present in low amounts.
Again, like the polyclonal versus monoclonal choice, it depends on what you need to do. But now you know the benefits and limitations of those things you can at least make an informed choice.
Has it been validated?
As we mentioned at the beginning, a good vendor will have done much of the antibody validation for you (although you still need to do your own!), but how much is enough?
In all cases we’ve ever seen, the antibody vendor will always include data on applications and species reactivity. That’s a given. But we think you should be able to see some of that data for yourself. Just saying “We tested this antibody in western blot,” isn’t very compelling. Instead, you should look for good testing data. What concentration did they use? Which species? What did the results look like?
Especially when it comes to selecting a new primary antibody, you want to arm yourself with as much validation data as you can. The Human Protein Atlas is a great starting place if you need to get an idea of what your staining should look like in a certain tissue or cell type if you have no idea what to expect.
We try to include as much data, from multiple applications, as we can about all the validation assays we perform. Hopefully, that gives you enough confidence in the antibody. If an antibody has been knockout (KO) validated in a research paper, we also include that information. You can also go and check on additional existing citations as well to see how an antibody performed in the hands of someone else.
There are always additional considerations, like the type of sample processing you’re going to subject tissue to, the conditions the antibody must bind under, the availability of certain antibodies, and even your budget. But the points detailed here should give you an excellent place to start from when it comes to choosing a primary antibody for your membrane proteins.
Did you also know we make everything in-house – everything comes straight from our lab to yours? You can also find the same protocols we use in the labs here that cover almost any immunoassays you might use They’re all absolutely free so go take a look.