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?

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You could try another source of protein other than BSA.

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How long do you let the blocking step go? Can you try letting it go overnight?

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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...

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Hi zoe8,
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.

regards
Hadrian

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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.

Good Luck!

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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?

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