Hello everyone! I am using BioID to detect some biotinylated proteins. I use streptavidin antibody, which is HRP conjugated. I have been experiencing background issues in general. I was blocking in 5% non-fat drymilk in PBS-T (0,001% Tween) and diluding my antibody in PBS-T with 1% BSA. Afterwards, a colleague found that milk powder contains biotin, so I switched my blocknig to 2%BSA in PBS-T, but now I get super dirty blot. Could anyone try to offer some insight to it? Thanks a lot
BSA should be fine, milk does have native biotin and as you found you get horrible results.
You can try TBS-T to see if you get better results.
The real key to good blots is wash lots, i often do 6 x or more.
Another tip is once you have your gel sandwich in clingfilm, spray some 70%IMS to remove static. This can give the impression of bad blots, when in reality you've got a good one.
I use SuperBlock (PBS) Blocking Buffer from ThermoFisher Scientific to block, then dilute the streptavidin antibody in TBS as Stanley Ng mentioned above. This process has been working fine for me.
My blot visualization issues come from the final step of timing the chemiluminescent substrates - being on too long or too short. Could that be something to look into?
Hey! Thanks for your answer! Well, being a light-sensitive procedure, I always made sure I did as fast as possible when it came to ECL. Most of the issues turned out to be with the blocking solution. I had good results with BSA 5% in PBS-T and blocking for 1 hour.
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