hello,
I am doing western blot for cancer cell and my protein ladder diffused during running electrophoresis and the sample is disappear. also it takes too much time more than 3 hr for migration. please help me I tried many time with different gel concentration 7% & 10% and I still have the same problem. And also I have problem that there is a leakage of the buffer from the chamber. how can I solve this problem?
If your chamber is leaking buffer, and your electrophoresis is also not working properly, I think it is likely that the electric current is following the same path as the leaking buffer (from the leaking chamber into the outer chamber) rather than passing through your gel and this is why your runs are failing.
There should be a rubber gasket which presses against the glass plates holding the gel, and seals the chamber so that buffer doesn't leak. Inspect the gasket carefully around its entire length. Is it twisted or bulging anywhere? If so, you might be able to re-seat it so it will seal, perhaps with a little bit of WD-40 or similar. If it is broken, nicked, or perished, you will need to replace it. Check the gaskets on both sides. Also, make sure that when you install the gels into the chamber, you are pushing them all the way down into the holder, and tightening any clamps properly. Practice setting up the chamber with dummy gels or buffer dams until you are sure you have fixed the problem with the seals. If you can't get it to stop leaking, you might need to replace some of your electrophoresis equipment.
Good luck :)
Problems happen only when you use this chamber? The question is because another thing to check is running-buffer. Are you sure it was prepared correctly? Have you tried to repeat electrophoresis in another chamber but using this same bottle of running-buffer?
Please post the buffer composition nad which loading buffer you use. Also post electrophoresis conditions and gel composition.
These answers are all relevant and on point--for example, if the buffer is 0.1X instead of 1X, that will affect the run time and focusing capabilities. If the buffer is leaking, that too will cause the same issues (and at face value, sounds like that may be the problem here). Also, be sure the gels are intact, that is, make sure there are no gaps at the spacer edges that will allow current flow out of the gel (because buffer is basically leaking that way), and that there are no obvious bubbles or empty spaces between the gel and the plates. Good luck!
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