Ever thought about using directly-labeled primary antibodies for your assay? You can buy them commercially, or you can label them yourself. With new kits and technology, it’s fairly easy to do.
Here are some reasons why you might want to add that label.
You can save time with directly-labeled primary antibodies
Sure, initially you will have to put in a little extra time to label your antibody. But companies sell kits that allow you to label antibodies in less than an hour. And once that is done, you will save time each and every time you use those antibodies.
The time-savings become particularly significant when you are talking about Western blots. Using directly-labeled primary antibodies, you will move from the first incubation step directly to the detection step. You’ll skip the second incubation step with secondary antibody (one hour) and the washes following the second incubation (~30 minutes). That’s an hour and half saved every time you do a Western.
You can multiplex with the same species of antibodies
Multiplexing, or the ability to detect two proteins simultaneously, is a huge benefit in fluorescence-based assays such as flow cytometry, immunofluorescence assays or fluorescent Western blotting. It can save you time, help you with normalization controls and make a qualitative assay quantitative.
The single most important requirement for multiplexing is making sure your two (or more) signals don’t erroneously overlap.
To solve this, most people use primary antibodies made in two different species of animals (say rabbit and mouse) and highly cross-adsorbed secondary antibodies labeled with different fluorochromes that specifically recognize the primary antibodies. This works great if your antibody choice is varied.
But what happens if your antibody choice is limited and you only have two antibodies from the same species of animal? You’re dead in the water unless you can discriminate between those two antibodies. Directly abeling antibodies will do just that. You can label one primary antibody with one fluorochrome and the second primary antibody with a different fluorochrome and go on your merry way.
You can clean up your assay with directly-labeled primary antibodies
Every time you use an antibody, it contributes background to your assay. And background makes your assay dirty. Sometimes it even makes it uninterpretable. Nobody wants that.
If you are using a primary antibody AND a secondary antibody, then your chances for background increase. If you use a directly-labeled primary antibody, you don’t have to worry about any background noise contributed by the secondary antibody. Ergo your assay is cleaner.
One antibody, many uses
If you use a primary antibody directly labeled with a fluorescent tag, then you have a powerful tool. Flow cytometry, IF, Westerns – you can do them all with the same antibody. Not only does it save you money, but it can be useful to have one antibody validated for a variety of uses.
You can keep it simple
Using a directly-labeled primary antibody keeps your assay simple. There are less steps involved so it decreases your chances of messing up. It gives you fewer steps to trouble shoot when things don’t go as planned. And there are fewer reagents to quality-control. Variability is reduced.
Overall, your experimental life is less complicated (it can’t help you with your personal life, sorry).
It’s not all good
Using a directly-labeled primary antibody can make your life easier, your assays simpler, your data prettier, and it can save you time.
But there can be some drawbacks to directly-labeled primaries:
- Your assay might be less sensitive without the added amplification step associated with the secondary antibody
- You need an antibody relatively free of contaminants and stabilizing proteins (such as BSA) before you can label it
- If your primary antibody amount is limited, you might not have enough to label
All-in-all though, directly labeled antibodies can help you out. Why not give them a try?
Photo courtesy of Hey Paul Studios.