For my ELISA, i am using PBS + 5% non fat dry milk as a blocking buffer. I have also tried using PBS + 1% Casein in TBS as a blocking buffer. I am still getting non specific binding to my polyvinyl chloride plates. Any suggestions for a better blocking buffer?
This document has been invaluable to me quite a few times when trying to identify suitable blockers for different ELISAs. Hope it's useful for you.
This document has been invaluable to me quite a few times when trying to identify suitable blockers for different ELISAs. Hope it's useful for you.
StartingBlock Buffer from Thermo Scientific works very well, and very quickly, no incubation needed.
Are you diluting your samples, conjugate...in the same blocking buffer? Sometimes its necessary not only to block the plates, but also to prevent nonspecific binding during each step.
I am using PBS (pH 7.2-7.4) with 1 % BSA (only V fraction) or with 1 % casein in my job and have not problems.
Claudia and Liubov, thanks for you input, I'm using HSA as my target protein so BSA would be too similar for me to use as a blocking buffer. I think I might have figured it all out be chaning concentrations of my blocking buffer.
I have also changed my SA-HRP concentration and that seems to have helped as well. That might have aided in my non specific binding.
Thanks for your response Gertrudis, i am diluting my samples in the same blocking buffer.
You could try gelatin, but in my hands both casein and milk are much better. Serum can be used, but it also contains albumin.
I would go with 1-3% BSA. Try to get ELISA [or RIA] grade..I have had trouble in the past with casein and dry milk. Both are contaminated with bacterial cell wall products, and in my case, bound anti-phosphoryl choline antibodies, obscuring the signal I wanted to read out..
Thanks for your response Gary. I wish i could use BSA but unfortunately i am working with HSA and they might be too similar.
Hi. We use 5% BSA or 10% FCS (expired batch) in PBS with 0,05% Tween for blocking in our lab. To diltue samples, we use half the concentration. But if BSA is not working for you, FCS might be problematic as well. I tried skimmed milk powder too, but I was not satisfied. Which plates do you use? I have good experience with Nunc MaxiSorp.
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