whole protein preparation for neutral pH HPLC

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

5 posts Page 1 of 1
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.
You may be interested in this webinar even though it deals with amino acids;

https://www.chromacademy.com/Amino-Acid ... Media.html
Jeff_11 wrote:
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.


Can you provide information on your lysate? Are you lysing bacterial pellets that you have used to express a known protein? Are you homogenizing organs and looking for total protein content? There are better ways to deal with certain lysates and that could help with the sample preparation.
Hi zoraku

Currently I'm lysing cultured mammalian cells (HeLa or HEK293) in a RIPA-like buffer (20mM Tris-HCL pH8, 10mM EDTA, 500mM LiCl, 0.5% lithium dodecyl sulfate, 5mM DTT).

I'm not confined to the above though.

What do I need:
Mammalian cells (any)
Whole proteins
Reduced protein
Protein complexes disassociated
Complex biological mixture
About 50-100ug

Any lysis or preparation methods are unimportant to me at this stage of method development. All I need is a process to get the sample sticking on the column and then work from there.
Jeff,

I would try precipitating the protein with ammonium sulfate, followed by centrifugation, then resuspend the pellet in a neutral buffer and see if that helps with the chromatography.
5 posts Page 1 of 1

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