whole protein preparation for neutral pH HPLC
Posted: Fri Feb 16, 2018 1:41 am
Hi folks
I'm just entering the world of HPLC and the first task I've been given is to develop a method for whole protein on a neutral pH RP-HPLC column.
Current conditions I'm using are:
Buffer A: 10mM Ammonium formate pH to 7-8 with Ammonium Hydroxide
Buffer B: 90% Acetonitrile, 10mM Ammonium formate pH 7-8
RP-4H proswift column
I have 2 questions:
1. I'm wondering what methods people recommend to prepare whole protein from total lysate?
Currently I'm precipitating protein from lysates on SP3 beads then eluting in 80% formic acid. Any suggestions for other means of sample prep?
2. Does anyone had success with this sort of chromatography under similar conditions?
i.e. the samples prepared as outline in Q1 don't seem to stick very well in neutral conditions- although it does stick in acidic conditions when using buffer A: 0.1% TFA, buffer B ACN 0.1% TFA.
Thanks in advance for any insights- I see many protocols for such RP-HPLC methods using peptides, but very few for whole protein.
I'm just entering the world of HPLC and the first task I've been given is to develop a method for whole protein on a neutral pH RP-HPLC column.
Current conditions I'm using are:
Buffer A: 10mM Ammonium formate pH to 7-8 with Ammonium Hydroxide
Buffer B: 90% Acetonitrile, 10mM Ammonium formate pH 7-8
RP-4H proswift column
I have 2 questions:
1. I'm wondering what methods people recommend to prepare whole protein from total lysate?
Currently I'm precipitating protein from lysates on SP3 beads then eluting in 80% formic acid. Any suggestions for other means of sample prep?
2. Does anyone had success with this sort of chromatography under similar conditions?
i.e. the samples prepared as outline in Q1 don't seem to stick very well in neutral conditions- although it does stick in acidic conditions when using buffer A: 0.1% TFA, buffer B ACN 0.1% TFA.
Thanks in advance for any insights- I see many protocols for such RP-HPLC methods using peptides, but very few for whole protein.