We have some antibodies that we always did the blocking and incubation in milk. We have recently switched to film based detection of the bands. However, when I use milk (5% in 1X TBS-T), I could not see Biotinylated Protein Ladder on the film. Is this a known problem.? And any tips to get around this problem are highly appreciated. Thanks in advance
see
Analytical Biochemistry
Volume 181, Issue 2, September 1989, Pages 318-320
no milk...use gelatin
Whilst most of the manufacturers recommend use of non-fat dried milk for blocking, this can sometimes be a problem for biotin based detection due to levels of endogenous biotin in the milk. I had this in the past, switching to 5% bovine serum albumin as the blocking agent resolved this for me. Alternatively try a different milk brand. It is possible it will have lower endogenous biotin.
Dr Harris and Dr Emmot, thanks a lot for your inputs. I am now clear that Milk intefers with biotin detection. I will switch to BSA for the antibodies that I used in milk previously. Much appreciate your time and effort.
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