Hello,
I'm currently testing some human sera on reactivity to specific proteins using western blot. We are expecting to find some reactivity in group of interest, but the problem is we see fairly high reactivity also in control group. Around 64% (total number 17) of them react to my proteins. The thing is I dont really believe this is a true reactivity but I dont know how to explain this.
Procedure is simple: isolated protein is loaded on gel, blotted on membrane, membrane is incubated with serum, then secondary antibodies against human IgG is used. The protein concentration used is around 10 ug/ul, sera are diluted 1:100, incubation overnight. Secondary antibody is against human IgG from goat. There is also possibility something is wrong with our control group, but we see really high reactivity even in group of interest (about 2-3 times higher than other studies suggest).
1. Avoid overnight antibody incubations. A 1h at RT should suffice.
2. Compare tests with and without primary side by side. Your non-specific signal may or may not partly be from the secondary
3. Optimize dilutions of both the human serum (primary) and secondary.
4. Make sure the membrane is properly blocked
5. Make sure the secondary is preadsorbed to human serum proteins (Min. X-reaction to human serum proteins).
Let me know how you get on.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining