I asked a question a while ago about improving phospho-specific antibody. Never got an answer but have been able to sort it out. Thought that I would share what I learnt with everyone else.
I had a background band at same size as my protein of interest. I was able to remove it using 2 methods
The first one involves making an acetone powder of the S to A , mutant, which the Ab shouldn't recognise, instructions for making this can be found in "antibodies, a laboratory manual by Harlow and Lane"
My second version is basically the same thing but slightly different. I prepared a large amount of SA protein extract. I diluted that to 1mg/ml in TBST and put it on a normal western blotting membrane for 1 hour. I then put my diluted antibody on that membrane. That way anything that recognises the SA protein will be removed. I then used the cleaned up antibody on my blot of interest.
Hope that helps anyone who is struggling with phospho-specific antibodies
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improving phospho-specific antibodies-how I did it
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