Secondary Antibodies


When using a primary mouse antibody on mouse tissues, there is more chance of getting non-specific background staining. For example the helicobacter picture below is crisp, brown only stains the bacteria. Whereas the Prostate cancer picture has brown all over the tissue. This brown stained prostate has non-specific background stain, it’s hard to tell what is positive for the KRAS antibody.

Human Helicobacter Pylori Human Prostate Cancer

To combat non-specific binding I use a kit from Vector Lab (M.O.M). Depending on the application you using (ABC, HRP, Fluorescent), you’ll want to pick the correct one for your target.

The secondary antibody use choose may or may not make a difference to you, but for me, I prefer a pre-mixed, ready to use version.

I tried the concentrated versions, every company seems to sell their own. Unless all of your employees have been trained by you from the ground up, then everyone has “their” own technique for everything. I noticed a big difference for concentrated versions with depending on who was running the assays. Some people like to use more than they should, for example (use 10ul – but really use 30ul), therefore we would go through much more than expected. Another difference I ran into with concentrated was the possibility of cross contamination. When I mix antibodies, I use a new pipet tip every time I dip into the concentrated stock. If new pipet tips are not used for every new dip, then the previous solution(s) contaminate every other solution it touches afterwards. So, for example, If I’m making 3 solutions of 1:100, 1:250 & 1:500 in PBST with horse serum, I would pipet 1ul of concentrated antibody up, then stick the tip into the first solution and draw/squirt multiple times to make sure all of it got it. Now if that same tip goes back into the stock solution antibody, the stock antibody would be considered contaminated. Even if those 3 runs (1:100, 1:250, 1:500) worked this time, chances are after the antibody is used again (another time) those reactions would not look the same or even work.

Therefore, to cut down on technique variability between people, I like to use ready to use secondary antibodies. And for those I’ve used several brands, Vector labs, and Enzo Life Sciences and others I don’t remember.

The Vector Labs ImmPRESS line is very good, nice dark staining, lasts for years (if stored properly) and comes with kits that help during the mixing process.

For Enzo, unfortunately their website is so complicated and hard to use, I can’t find the product to reference to.