We have a protein at high concentrations of urea to maintain its solubility. When we perform a Bradford assay, the concentration is underestimated due to protein aggregation. Is there an assay/procedure to quantify protein concentration on low solubility proteins?
Urea is usually pretty good at disaggregating proteins, but for more powerful disaggregating try guanidine-HCL or guanidine isothiocyanate.
there are plenty of reagents which can be used even with the High Concentrations of Urea or GnHCL. I have always used Bradford of Sigma with GNHCL. if this is still giving problems, one can actually use SDS PAGE as analytical tool to do quantification by Densitometry using silver staining procedure. this i know is tedious but is a option when no other option is available. There are people who use HPLC also to do a similar quantification.
In my opinion, a much better way to measure protein concentration is to use intrinsic UV absorbance at 280nm, which entirely avoids the problem of dye binding variation. This works really well in 6M guanidine hydrochloride, which will dissolve most aggregated proteins. You could also try resuspending the protein in 10mM NaOH (pH11) or 10 mM HCl (pH2), and measure by UV absorption (dyes will change their characteristics in these pH values). Make sure you centrifuge the preparation at > 13 000 g to remove the aggregate and reduce light scattering, which can provide erroneously enhanced values. Sadly you will have to get the protein into solution to accurately measure it, unless of course, you can precipitate it without salts, dry it and measure the mass with a very good balance... I hope this helps.
Thank you all for your help I will consider your suggestions. Originally, we wanted to employ Bradford since the extinction coefficient of our protein is too small and after conjugation w/ a fluorescent dye molecule (lambda max ~ 546 nm )the fluorescent dye absorbs somewhat at 280 nm, leading to potential errors in the fluorescent dye labeling efficiency determination.
If you know the labeling stoichiometry with your dye, then the absorbance of the dye and its extinction coefficient can be used to quantify the protein. The easiest way to measure the stoichiometry is by whole-protein mass spec. The extinction coefficient of the dye is probably given by the vendor.
Another approach might be to precipitate the protein (acetone?), redissolve the in dilute NaOH solution (which usually will redissolve most proteins completely, and do a biuret protein assay.
I agree with Karl. Acetone is an option, I personally prefer TCA or TCA/acetone for precipitation. Probably lowering your urea concentration beforehand would help. TCA / biuret is an option but TCA / BCA or simply TCA/ bradford are also possible.
Here are some protein precipitation protocols. Check the TCA MSDS before use.
http://wolfson.huji.ac.il/purification/Protocols/ProteinPrecipitation.html
Just another suggestion... if you have problems with other methods, you can try QUBIT (http://www.lifetechnologies.com/order/catalog/product/Q33212). If you know somebody with this kit, ask to give it a try! It works really well for most proteins and in a very wide range of conditions!
Cheers
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