By Marina on Wednesday, July 7, 2004 - 04:53 am:

Hello! I am new to protein research and we are trying to profile the proteins of cell and tissue lyzates. Working with C18 (luna from phenomenex) I just got kind of smear, which I thought might be because superimposed peaks or may be the proteins are just precipitated on the column? Now we are going to try 2 dimensional HPLC with strong cation exchanger in 1st dimension (PolySULFOETHYL Aspartamide 300 A). We intend to collect fractions and inject them into LC-MS (C18). Are we on the right way? What mobile phase should we use with the cation exchanger? What percentage of ACN should it contain so the proteins are not precipitated? Why everyone use protein digests with 2 dimensional HPLC and whole proteins working with rotofor + HPLC? I will be very thankful for any advise and sorry for the long message.

-------------------------------------------------------------------------------------------------------
By HW Mueller on Thursday, July 8, 2004 - 12:13 am:

You are brave! I have a hell of problems with "single" proteins.
Check "proteomics" to get some info (literature, internet, companies like Bio-Rad, etc.).Also, a book on protein purification may help to learn how to handle proteins.
You usually need a mobile phase gradient when you want to do proteins on RP (usually a highly pre-purified fraction is used here).
Precipitation problems relating to mobile phase are easily checked by adding, dropwise, sample to be injected to some mobile phase in a test tube and observing carefully.
Small peptides are usually more easily chromatographed than most proteins.
Maybe someone else dares to give more details. My experience relates to proteins that have already "seen" many separation steps at pharmaceutical companies before I get my hands on them.
What is rotofor? If this has to do with electrophoresis the answer is clear: You can split up the complex soup into thousands of fractions with two dimensional electrophoresis.

-------------------------------------------------------------------------------------------------------
By Marina on Friday, July 9, 2004 - 12:22 am:

Hello! Thank you so much for your answer! I will show it to my supervisors so they see it is not just me. Rotofor is an instrument sold by Bio-Rad which separates the proteins into 20 fractions by isoelectric focusing as in electrophoresis, but the fractions are liquid and nod adsorbed to gel and so it can be further separated by HPLC/MS. This instrument is very expensive and we want to try doing kind of same thing with Cation-exchanger. I will try to find out proper ACN concentration by mixing in the test tube. Thank you for the advice, and I will be glad if there are any other suggestions.