I was doing SDS-PAGE when i ran the dye started to seperate into two layers. i dont know why. the pH of the buffer and volt everything were ok. i checked everything buffer, pH, and also made the dye freshly. but it started coming like this again. i have attached the pic of my gel.
can anyone tell me how to troubleshoot it?
What about your gels that you are making? Are they prepared in exact concentration? Mostly the smear stuff happens if the pH of the buffer is not good. Also, the laemmli buffer that you make does it has a proper pH? What about the buffer that you are using to isolate your samples. I hope this provides you with new troubleshooting options.
Hi Brindha, in addition to Mayank's answer, the different color you are seeing in the bromophenol blue dye front can be indicating that your samples are too acidic, too salty, or both.
Is the far left lane your MW standards in SDS sample buffer and the rest of the gel your samples in SDS sample buffer?
Then I am inclined to say that say that your samples are the problem. A quick experiment to do would be to do a 2x serial dilution of your sample in the SDS sample buffer and run it.
If the problem is with your samples, then you should see a gradient.
I suggest you to check the pH of your loading dye as well as your extraction buffer (I mean the Buffer you have used to extract and dissolve your protein). . If your sample is extract then use less concentration make a dilution then load into the gel.
So you try adjusting the pH of your loading dye using 1M Tris-Cl.
I hope it will help you
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