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- Posts: 33
- Joined: Fri Nov 17, 2006 4:50 am
- Location: Australia
I have to quantitate very low concentrations (<10 ug/mL) of a peptide (~4kD) and a protein (~55KD) in a solution.
Ideally one HPLC method would of been best, however...
The protein crashes out in the higher organic component of the mobile phase when using reverse phase chromatography.
So I have since developed an SEC method for the protein, and a RP method for the peptide using a Luna C8 (3 µm 100x2 mm 100 Å) column.
The Problem
I need to separate the peptide from the protein for this analysis. Using a centrifugal filtration method, the peptide aggregates to the protein, and nothing comes through. So, I was wondering if experimenting with varied amounts of SDS in the sample to prevent aggregation and then using an Amicon with a 10KD cutoff. And then using RP on the filtrate, for the peptide quantitation. But I am not sure of the highest concentration of SDS I can use without it causing damage/if any to the column. The column will be dedicated to this method.
I am not even sure if the SDS will prevent the aggregation of the peptide with the protein. I can only try. Not being a protein chemist, I feel I am doing this blindly. This whole project has been very challenging, I now have a deep respect for protein chemists.
One other thing, I have just read that there are these guard columns you can attach to your column that can remove SDS from your sample. Has anybody used these?