Non-specific binding in binding assay - BSA blocking step (Jul/31/2005 )
I'm having trouble preventing NSB in my binding assays - at the moment I use 0.5% BSA / 0.1% Tween 20 in PBS but I'm still getting greater than 50% NSB. I can't use skim milk powder as it contains one of the proteins I'm looking at. Any suggestions as to what I can do to improve my assay?
You could try another source of protein other than BSA.
How long do you let the blocking step go? Can you try letting it go overnight?
I usually block for 2 hours at RT. I guess I will have to try increasing the amt of BSA / Tween and/or the time...
I always use 5% BSA in TBS instate of PBS. No Tween 20 in my blocking step. I usually bolck for one hour at room temperature.
Try it out.
Hi, try using 5% BSA and 3% Tween20 in PBS. Works usually good for me. Or, instead of BSA, some people use gelatine or slim fast.
you said that you couldn't use bovine milk to block as it contained one of your proteins. This isn't in bovine serum as well?