Chromatography Forum

Hi,

I'm looking to develop an HPLC method to quantitate total protein in 4 different manufacturing column buffers. We expect very low protein in each solution (<3ug/ml). The first solution has the greatest chance of containing detectable protein, which could be composed of a range of our expected product and then carryover from fermentation, including small peptides. With each column, we expect less protein but need an assay with a low quantitation limit to show this.

I've heard that there might be a formic acid (or similar) way to pretreat the samples and then run on reversed phase or SEC to be able to quantitate (i.e., integrate the whole chromatogram). Is anyone familiar with this or another method that could be adapted to measure total protein? We've tried more traditional BCA methods, but those are problematic for various reasons so perhaps a RP or SEC method is a good possibility.

Thanks,
Dave

What´s BCA?

BCA = bicinchoninic acid method

Never heard of it, also havn´t heard of using HPLC for the determination of total protein. How about Cu complexing (Biuret, later Lowry...) or Coomassi blue methods, or just UV or Fluorescence. The solutions may have to be concentrated. If you do a search in a library or the internet you will be inundated with info.

BCA is a Cu assay with a readout at 562nm. It has inherent buffer interference issues at the concentration levels were are going after (< 3ug/ml). That is why I though an HPLC method might work.