Hello everyone, I'm now working on ELISA. I'm testing TNFa on the serum of pigs.
The results are pretty inconsistent. I did the 1st plate and everything came out well. But I'm now running a second plate but the signal of my samples is very light blue while the stand came out blue. The OD of my standard (2000 pg/ml) is around 3, is this too high? and the OD for my samples most of them are
When preparing your sample did you add protease inhibitors? How were your samples stored?
Hello Robert Adolf Brinzer
I store my sample in -80 C and I didn’t add protease inhibitor. I use 100 ul of my samples and directly add them into the wells without diluting them.
Sometimes proteases in the sample can degrade the protein of interest or degrade the antibodies in the ELISA. Adding a protease inhibitor cocktail can sometimes help. How long were the samples stored between running the two plates?
Robert Adolf Brinzer Thank you for your recommendation. By the way, my first plate is fine without using a protease inhibitor, so I'm now curious that would it be possible that some of my reagents are not in good condition. I stored my samples for a few months before running ELISA. By the way, between these 2 plates, they are different tubes of samples but they are from the same day that I collect just from the different pigs.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays