I have recently got some problem with my 2-D gels. Please check the attached gel pics for details. I have used 2-D clean-up kit (Biorad) to remove the impurities from the sample. I think there should be some problem with the second dimension. But I have tried to run several gels at one time with different concentrations of SDS (used upto 0.6% SDS in the running buffer for second dimension). The uploaded 2D gel picture with tear fall effect was run with 1xTris-Glycine buffer (0.1% SDS). When I have increased the SDS concentration to 0.6%, the “tear fall effect” got decreased (but at the higher molecular weight region it still persists). I am also uploading an old gel for reference. For running old gel, I have used only 0.1% SDS in running buffer. So I wonder from where this problem came suddenly?.
Procedure used for the run
17cm IPG strips, pH 4-7.
Protein samples isolated from PBMCs
Loading concentration used 800 microgram.(Protein samples were prepared using 2 D clean up kit from Biorad)
Equilibration buffer used with 2% SDS for 45 minutes
Running buffer used -Laemlli SDS electrophoresis buffer (25mM Tris base, 192mM glycine, 0.2% SDS). To rectify the problem, I have increased the SDS concentration upto 0.6%.
Instrument used for second dimension PROTEAN II system (biorad).
Second dimensional running conditions- 85V (constant), 20Hrs.
Hello,
Do you have anode buffer and cathode buffer separated in your Protean II system ? If yes, you may have a leak of the upper buffer, which make a depletion of SDS in the upper buffer (cathode) during the migration. I had exactly the same problems with my electrophoresis unit (not a Biorad System) : there were leaks from my cathode buffer chamber. After changing it, it was OK.
thank you Pionneau for your valuable suggestions. Our instrument has separate cathode and anode buffer tanks.I also checked for leakage issue. Any other possibility?.
Could it be a problem with your water in the buffer ? Do you use ultra-pure water ? Maybe the purification pack of your water purification system is too old and need to be replaced.
@Pionneau,
Yesterday i tried to run the gels with 3X concentration of running buffer (cathode buffer), but results were again unsatisfactory. Is it something to do with Iodoacetamide solution?. I have changed the bottle recently. If it so, why only higher molecular proteins are get affected?.
Quality of ammonium persulphate (APS) may also affect the electrophoretic separation of protein. always use ultrapure grade and it is recommended to use it freshly prepared on each day of run. this may help you.
You should try to follow the standard protocols as an starting point. Then I suggest you try with new acrylamide, APS and TEMED bottles, also doing fresh TRIS-HCL solution checking carefully the pH (this is an important point). Urea and water quality is also important. Check also that all is right in the equilibration steps and the temperature during the electrophoresis.
Your old gel pic looks okay except some abundant protein
BioRad sample cleanup kit or acetone precipitation gives same results. (but use good acetone and keep it in deep freezer)
this kind of picture can be due to problem with sample buffer and protease inhibitors. and old stock of running buffers
But first correct anything which you may have changed inadvertantly from previos experiment.
Changing the parameter like equibrium time and other gel runnig conditions usually are not serve much purpose
You may start afresh with standard starter kit protien and see if it gives similar problem.
- Badges
- Science topic