help!!! biorad assay vs. BCA assay

ckcassidy at ibt.tamu.edu ckcassidy at ibt.tamu.edu
Mon Jun 16 15:36:21 EST 2003

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=0


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