Protein quantification - HPLC

[agill]

Hi
I am looking for some advice/help while trying to develop a method for roughly quantifying proteins that are secreted into a fermentation broth by certain yeast strains. This will be a general protocol that could be used for assessing levels of certain proteins that are secreted into the medium by cells making them. In particular I am looking for the ability to be able to screen the spent culture medium after growth of desired strains. I want to be able to directly load spent culture supernatant onto a column, and then roughly compare the relative levels of protein produced by different strains, based on peak area of the protein of interest. I could establish standards for the protein of interest by purifying the protein and loading known quantities of it on the column and calculating peak area corresponding to different protein concentrations.
I am wondering whether there is any tried and tested HPLC method that someone could direct me towards. Would it be better to use SEC-HPLC, or RP-HPLC (with a C4 or C8 column). Does anyone have experience in developing such a method ? If yes, could you share specifications of columns that you might have tried.
Based on the specific source organism, the protein of interest that I am looking at is in the size range of 40-55 Kilodaltons.
Any advice is greatly appreciated.
Best regards

[tom jupille]

There is no single answer to such a general question. What is important is how the protein(s) you are interested in differ from other proteins that may be present. Then, you pick a technique which is selective for that kind of difference.

So, if other proteins are likely to be different in size, then SEC would be a good choice. If your protein is significantly more (or less) hydrophobic than other proteins, and you don't mind denaturing the protein, then reversed phase would be a good choice (although, at 45 kDa, you're getting to the ragged edge of what RP can do). If you need to retain biological activity, then HIC may be your best bet. If it has a relatively high (or low) pI, then ion exchange might be indicated. If your protein has a specific interaction with a particular substrate, then perhaps affinity chromatography would be the best solution.

As a general starting point for a 45 kDa protein, I'd look at ion exchange.

[Kostas Petritis]

Unless you have a very small amount of secreted proteins, you will have separation problems (i.e. protein coelution).

There are methods that involve protein digestion, use of isotopic labeled peptides and analysis by LC-MS, but I won't elaborate as that is not what you were asking...