2D gels: Is there a protein assay thiourea is compatible with? - proteomics (May/25/2007 )
Dear all!
I was wondering if anyone had any experience with using thiourea in a protein assay? I am currently trying to prepare my protein samples for running on the first dimension of a 2D gel. As they are membrane proteins it was suggested that I add thiourea to the rehydration buffer, however, it appears thiourea isn't compatible with the BCA assay. Does anyone have another suggestion of a protein assay that maybe compatible with thourea?
Alternatively, has anyone tried to do 2D gels on membrane proteins? I've read that its particularly tricky to do this.
Thanks for your help 
I was wondering if anyone had any experience with using thiourea in a protein assay? I am currently trying to prepare my protein samples for running on the first dimension of a 2D gel. As they are membrane proteins it was suggested that I add thiourea to the rehydration buffer, however, it appears thiourea isn't compatible with the BCA assay. Does anyone have another suggestion of a protein assay that maybe compatible with thourea?
Alternatively, has anyone tried to do 2D gels on membrane proteins? I've read that its particularly tricky to do this.
Thanks for your help
it is tricky especially if the membrane proteins >100 kDa; try rehydration of strips (incubation with protein solution) O.N.; use maximum loading capacity of proteins and protease inhibitors;
protein determination with UV-radiation may not be affected by thiourea
We routinely use the protocol below for 2D PAGE samples. It is a modified Bradford Microplate assay, and works very well. It gives linear response over the range 0 to 2.5ug protein. Don't forget to allow for dilution factors when you calculate your final protein concentrations!
Alternately, you can try the BioRad Experion instrument - works well, as long as your 2D buffer is diluted at least 1:10 (and your ladder standards have a similar amount of urea in them).
UV will not work - thiourea and urea absorb strongly in the blue region.
As for membrane proteins - try detergents C7BzO or ASB-14 - both very good.
Good luck!
Important things for the assay;
1. Do not change the volumes - they are crucial.
2. Whatever you do, the samples and standards must be in an identical buffer - Bradford is very sensitive to salts, pH etc.
3. Vortex every sample between dilutions to ensure they are accurate
Procedure
1. Make serial (1:2) dilutions of a BSA standard covering the range 0-0.5mg/ml in 2D PAGE buffer (i.e. we start with 2mg/ml BSA, and take 100ul into a further 100ul of 2D buffer = 1mg/ml. And so on – 8 tubes altogether, down to 0mg/ml). Standards can be aliquoted, frozen and stored.
2. Make dilutions of your sample as required IN THE SAME BUFFER AS THE STANDARDS.
3. Add 5µl of each standard (0 to 1mg/ml), and 5µl of each sample to microtitre plate wells - we always do everything in triplicate.
4. Dilute 1 part Bradford reagent concentrate (BioRad – cat# 500-0006) with 4 parts MilliQ H2O.
5. Add 300µl Bradford reagent to each well. Incubate at room temp 15 mins (no shaking required).
6. Read absorbance at 595nm. Absorbance must be read within 45 minutes.
7. Quantify your protein according to your standard curve.
I was wondering if anyone had any experience with using thiourea in a protein assay? I am currently trying to prepare my protein samples for running on the first dimension of a 2D gel. As they are membrane proteins it was suggested that I add thiourea to the rehydration buffer, however, it appears thiourea isn't compatible with the BCA assay. Does anyone have another suggestion of a protein assay that maybe compatible with thourea?
Alternatively, has anyone tried to do 2D gels on membrane proteins? I've read that its particularly tricky to do this.
Thanks for your help
it is tricky especially if the membrane proteins >100 kDa; try rehydration of strips (incubation with protein solution) O.N.; use maximum loading capacity of proteins and protease inhibitors;
protein determination with UV-radiation may not be affected by thiourea