I have repeated the western for several times, but it shows the same problem. It seems that some bands are squeezed(2 and 5 in the first picture, 2 and 4 in the second picture). I use the commercial Bio-rad gel and firstly I thought it maybe the problem that the comb was not well removed so the well was broken. However, last time I do it very careful and verify there's no problem with the gel, it still show like this. When the electrophoresis starts, I can see the exactly which sample is squeezed through the loading buffer. Last time, I found the well 4 and 5 for two different times are from the same sample, well 2 is also the same. That means it maybe the problem of this two sample, but I can't get the point and the exact reason. Does anybody encounter the same problem like me? Hope to see your suggestions.
PS: 1. Samples are 30ug/well and 15ul/ well. Has run two times with the same sample and get the same problem.
2. Other people in our lab using the same western system can get straight and good result.
3. For protein extraction, I scratch the cells in PBS, centrifuge to collect cells and add with RIPA lysis. Would the different residual of PBS affect the salt concentration in the samples, which I have heard may affect the electrophoresis.
Thank you all.
Hi Shirley,
I would suggest there could be two factors you could address:
I hope this information helps, good luck with your experiments.
Yours sincerely,
Brandan
Hi Shirley,
If I interpret your point 1 correctly; you have 30 micrograms per well? That's a lot of protein. Attached is a SDS-profile of a BSA standard with 5, 4, 3, 2, 1 and 0.5 microgram of protein. In particular, western blot is very sensitive and should not need that much protein. Perhaps you overloaded your gel?
The concentration of salt does affect the migration pattern. I know this because sample that contained higher level of salt (difference in 1 M) migrated at different rate. Since you are lysing in PBS, I don't think there would be a huge variability between every sample.
Is the squeezing only occuring for a particular sample?
Thank you all. I just suspected it's the problem of a particular sample. All other conditions are used very well by my colleagues and they can get good band.
Maybe try another protein extraction of the particular sample (if you can), there could just be degradation or another problem in those samples.
Starting again sometimes allows you to find if there was any mistakes made in protein extraction, or to narrow down where the problem lies when running the gel etc.
I hope that these tips will help.
Maybe you can desalt your samples on G-25 column or by dialysis and then see what happens. Be sure to check if your proteins are soluble in solution with less salt. Also like Derrick said try reducing protein concentration per well.
Good luck.
Hi Shirley,
On first glance it looks to me like there is uneven protein loading (the lanes with more protein will become more borad, and the ones with less protein will be squeezed),do you quantify your protein amount before loading? (Did you mean 30µg or µl?) Especially if the same sample results in the same problem, this could be the case. My suggestion would be to measure protein content of your samples and make sure you load similar µg per well.
Another thing I can suggest is to scrape cells directly in the RIPA buffer after repeated washing with PBS. It works very well for me (e.g. 180-250µl when using 3.5cm dishes), and it may help keep your samples more similar, but depends of course on how well your cells like to adhere.
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