I need to confirm some results of 2D immunoblotting by carrying out the western blot (1 D) using same samples that are prepared for 2D using rehydration buffer contains 8M urea, 4% CAPS, 40 mM trisCl 6.8 and DTT. as i know that boiling urea will cause carbamylation. i wonder whether i can use the sample directly in my WB without boiling.
when i perform the 2 D gel of my proteins in the rehydration samples the second dimension works perfectly so i assume loading the samples directly will also work.
is the equilibration step after IEF contribute to the resolution of the 2nd dimension?
Hi Nashed
For western blot, If you have added urea, no need of boiling. But If possible, avoid the urea for WB sample preparation.
Yes, equilibration plays very crucial role of the resolution of the 2nd dimension after IEF.
No problem at all, at least it is our experience, so go ahead. Proteins must be solubilized and heating helps. I do not think carbamylation is an obstacle for western blot and it should not change the results.
Boiling is not necessary with urea. CHAPS may reduce performance of Western Blot but not urea. If you dilute enough your sample in loading buffer, you will not have any problem.
I heat the samples and I have no probem. I had a zig-zag effect on mu 1D-gel, but it came out from too much different volumes of my samples. I precipitated them with TCA/acetone. But when the sample volumes are quite equal I rather have no problem. good dillution with loading buffer can help.
thank you all for your replies. here what i found.
First, I tried to load samples directly without adding loading buffer ( SDS, mercaptoethanol..) assuming that 2 D rehydration buffer has enough denaturing power . the results were bad with smearing and some zig-zag pattern. i repeated with adding SDS loading buffer without heating. the results improved alot but i found some precipitation indicated by proteins on the stacking well. i repeated again with 5 minutes centrifugation, the results were perfect.
Hi Abdullateef,
incubation at room temperature with SDS and b-mercaptoethanol for 15-20 min helps a lot. I run my samples that were extracted in urea.
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