I am facing problem with cleavage of a protein using hydroxylamine. I used guanidine HCl and K2CO3 and then hydroxylamine pH 9 with a 45°C temperature for 5 hrs. After the reaction I got the protein precipitate. I used BSA for control and couldn't find any result in the SDS-PAGE. Then I used only hydroxylamine HCl pH 9 with trizma 0.1M, and digested for 4 hrs. This time I didn't observe any precipitate after the digestion. However, after desalting with Bio gel, and precipitation using trichloroacetic acid and acetone, I ran it on a SDS-PAGE, but bands aren't that clear. Please suggest a possible solution.
Are you sure your protein will precipitate once digested? which is its molecular weight? can you skip the desalting/precipitation step and run the gel directly after digestion? which protein:NH2OH ratio are you using?
Thanks for the reply. I have used BSA for control. And the concentration is 5 mg/ml in reaction buffer.Final buffer for cleavage is 2 M NH2OH, 0.2 M K2CO3, 3 M GuHCl, pH 9.0. After 5 hrs at 45C I got precipitates. Even if I skip desalting/ further precipitation after that, it is difficult to load as there are presence of precipitates already. But when I used only 2M NH2OH at 5 mg/ml concentration, pH 9. I got clear solution after digestion. But when I ran it on gel after TCA desalting/precipitation, I can see bands but not at all clear.....
Could you skip the desalting/precipitation steps even in the 2nd case and see what happens? can you run a MALDI-ToF on your lysates?
I dont have MALDI-ToF facilities here. but I have tried to load samples without desalting, could not get any clear bands in gel. But there are very very light bands and smear in the expected regions. but that is not convenient.
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