Millipore has come up with an instrument based on infra red technology that might help. It uses neat sample, max 2microlitre volume.

I asked about this instrument, but it's necessary that the sample dries and with glycerol is impossible. Thank you for your help.

you can use BCA kit

You can measure the OD of the protein at 280 nm. Glycerol will not interfere with your measurement. However, you are very likely to have pipetting errors with 50 % glycerol in your protein solution. So be careful and make multiple measurements.

Thank you for your help.

You can use amino acid analysis. This will give you an exact estimate of the protein concentration.

It is impossible to dilute the sample, because another problem is the low protein concentration.

Thank you for all your answers.

Try 2-D Quant Kit (Amersham). The company tested the reaction against glycerol - though 30% (w/v) - for assay compatibility (with no interference). Besides, as the assay (based on precipitation) has a linear response (with negative slope) to protein in the range of 0–50 μg, it might be good for your diluted samples.

Good luck!

We use Pierce (Now Thermo) 660 nm Protein Assay - this should be compatible with 50% glycerol according to instruction sheet. Linear range is between 50 and 2000 ug/ml, but quantification is still possible below 50 ug/ml (albeit no linear curve).

Alternatively, but significantly more work, run an SDS-PAGE gel and stain with colloidal coomassie, and quantify by densitometry. This will also give you some information on protein purity.

You can simply dialyze the protein solution using a buffer of your interest overnight, then quantitate the protein with the BCA kit. This method will also concentrate your protein, because glycerol leave the bag with a consideribly amout of water.

Thank you for all your answers. I'll try it when I come back from my summer holidays.

Hi, what seemed to work for you? I have the same problem...

I'm using Bradford now, but I don't know if the results are very accurate.

If you have plenty of sample, I suggest performing a "buffer exchange" using an Amicon 3K MWCO spin filter on a portion of your sample. This will get rid of the glycerol and you will be able to perform a BCA or Coomassie protein assay on your sample with greater accuracy. If you have very little sample, then I'd suggest dilution (10X if possible), and then make your standards and blank in a 5% Glycerol solution - this would adjust for the matrix effect of the glycerol.

I have the same problem with a 25% glycerol buffer  ... Did you find any reliable method? Thanks in advance

I did not find any good method. We now measeure the protein content by densitometry with an SDS gel running several lanes with a protein of known concentration. But it is a very time consuming method and you need a protein of a similar molecular weight to compare.

To quantify protein in any buffer, this is a great kit: EZQ Protein Assay (https://www.thermofisher.com/order/catalog/product/R33200). You basically spot your protein of interest on paper, wash off the salts, detergents etc and then measure protein concentration. I've used it to quantify proteins in 8 M urea, 3% SDS, 20% glycerol...

Thank you Vikram, I don't know this kit,but it seems quite good option. However, I am not sure if 50% glycerol could be dry properly. I would try.