I am facing the a problem in my SDS page gel. protein marker and samples not get well separated. All samples were stop separating at the middle of separating gel (protein marker also). Dye front went to the bottom. But after staining, I can see only protein separation up to middle of the separating gel.What would be the reason for poor separation of the protein. I checked the power supply and SDS-PAGE system. No damage to the wire.
Usually, this happens when the pH of stacking and resolving gel buffers is not correct. Did you use freshly made gel? Also, make sure your buffers for stacking and resolving gels have correct pH.
Yep. I use freshly prepared gels. Reagents were worked fine before. I am using the same protocol. nothing changed. But it didn't work after some time about 3 weeks. I checked the pH of Tris buffers. They are Ok.
Is there enough of SDS in the loading buffer? Also vortex your loading buffer, because while storing in refrigerator, 6x SDS will crystallize, and make sure to use it when warmed on room temperature. Looks like something is wrong with the charge of your protein if it stops separating and dye reaches the bottom.
I completely agree with Barbara Umek. I want to add some more points...
1. what concentration of separating gel you have used.
2. After adding of protein loading dye to the protein samples, how much time you have kept at 100C for protein denaturation.
what concentration of TrisCL, SDS, Glycine, you are using and check the pH of running buffer also.
Hope it will work...
Good Luck
There are many factors that affect protein separation on SDS electrophoresis some result from preparation of protein sample or sample buffers and nature of the proteins as well as run condition .make sure there are no bubbles are present in the gel these will hinder movements also check protein concentration because portion of the concentrated protein precipitated during the stacking process . you might try to increase voltage by 25 percent during stacking. Try to run a known standard protein in the same SDS electrophoresis condition if it works then the problem with your protein preparation
good luck
I agree with Hunaiti. I found that if not properly preparing my samples has caused issues with my Westerns. I also agree that the quality of your gels could be a potential issue. What are the percents for the stacking and resolving. One issue we had a few weeks ago, is our SDS was contaminated and our Westerns were not coming out right. If you think it is the voltage, you can look for bubbles to make sure the current is running.
Hope that helps!
Running buffer can also be the problem. I would recommend making fresh buffers and gels with special attention to salt concentration and pH and retry running. If your marker is a commercial one with dye already added, and is running the same as your samples, it's unlikely the problem comes from sample preparation.
I agree with Barbara U. Does your upper reservoir buffer contain SDS? As proteins migrate in the E field they will continually shed C12-sulfate anions as the anions migrate faster than the SDS-protein complex. Steady state of SDS anions leaving the complex and being replenished by anions migrating down from the reservoir buffer. Without replenishing anions, the protein migration slows & nearly stops. The original Laemmli system called for 0.4% SDS in the upper reservoir buffer, although that cooncentration is actually overkill.
I agree with the answers above. The most common reasons for problem you described in my experience besides sample preparation has been suboptimal pH of the stacking or resolving buffers or a problem with the running buffer (often due to reuse).
I agree with you, my experience said that Run time of your SDS not take more then 3 hrs, then it not separate perfectly . For that you have to concentrate on your SDS gel preparation and sample preparation. also your electrode buffer should be freshly prepared.
Hello Bhagya.. I got the exact same problem now. Did you find out what was the issue?
- Badges
- Science method