I need to measure the protein concentration of lipid raft isolated by density gradient centrifugation. Proteins are separated across a 25%, 20%, 15%, 10%, 5% and 0% Optiprep gradient. I collect fractions across these concentrations and would like to do a BCA protein assay for all the fractions to see the protein distribution across the fractions. My initial BCA assay showed protein amounts way below what was published (Macdonald & Pike. A Simplified Method for the Preparation of Detergent-free Lipid Rafts (2005). J Lipid Res 46:1061). I found from a Google search reports of Optiprep reducing absorbance values in protein assay. So I added the increasing percentages of Optiprep to a known concentration of protein, expecting to find high Optiprep concentration reduce absorbance values. To my surprise, there was no change in absorbance values in the presence of Optiprep. Before I repeat this assay, can anyone tell me does Optiprep reduce absorbance values in a protein assay? Thanks.
According to this document, Optiprep (iodixanol) is compatible with dye-binding assays (i.e. Bradford, amidoblack), but not Folin reagent-based assay (i.e Lowry) unless the iodixanol concentration is under 5%. The Bradford assay is recommended
http://www.axis-shield-density-gradient-media.com/S09.pdf
Hi thanks for that document. I am expecting low protein concentration so am wanting to use the BCA assay, which I assume has a working range which covers a lower concentration than Bradford. I'm intrigued by table 4 of the document as it reflects the buffer I use- I do have 0.25M in my buffer. Is that table saying increasing amounts of Optiprep actually increases the absorbance?
Table 4 shows that iodixanol (Optiprep) has an absorbance at 340 nm. This absorbance would not interfere with the BCA or Bradford assay, which measure at 562 and 595 nm, respectively.
The Bradford assay and BCA assay have about the same sensitivity. In my experience, using BSA as standard and commercially prepared Bradford reagent, the Bradford assay can measure 0.5 µg of protein in a 10 µl sample (0.05 mg/ml) quite satisfactorily, using 200 µl of Bradford reagent in a 96-well plate (the pathlength is about 0.5 cm). There is room to improve the sensitivity of this convenient method by using a 1 cm pathlength cuvette and increasing the volume of sample. I would expect to be able to get to 0.01 mg/ml or less if necessary.
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