The Biorad and BCA assays are relative. Another option you have it so use the extinction coefficients. http://us.expasy.org/tools/protparam.html will give you those, but the most accurate way to find these are listed in a paper by Nick Pace from Texas A&M “How to measure and predict the molar absorption coefficient of a protein” Protein Science 1995). You will never really be any better than 10% of the real value. The rest of this information is anecdotal: When a protein is not properly folded high salt (>200mM) will tend to force the protein to the pellet. High volume and low salt works best to keep the protein soluble. Once purified (HIS, S-Tag, GST, Etc…) the levels of protein can be increased. I have found that after column chromatography it is best to dilute the sample 50% prior to dialysis. Proteins that aggregate are best dilute. Once, pure and in high salt they can be concentrated. As for pH, make sure you are at least 1 pH unit from the PI. If the PI is 7, then you need to keep the ph under 6 or over 8.
You may want to read a paper by Rachel Kapust in protein science 1999 8:1668-1673. talking about fusion tags for protein solubility. I have had good luck with MBP and Trx. Limited luck with GFP, S-tag and GST.
selphydeg at yahoo.com wrote:
> I am working on a thermophilic protein overexpressed in E.coli. The first step
> in purification is heating. I used Biorad and BCA assay to determine the
> protein concentration, but the result from the two assay is very different.
> Protein concentration determined by Biorad is 2X higher. I also have trouble
> purify the protein, and concentrate the protein to more than 1.5mg/ml. Does
> this all point to misfolded protein?
>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0http://biowww.net/mynews/tree.php?group_name=bionet.molbio.proteins&begin=0
