I have been doing Western Blot for about 4 years in my lab and now some samples are not running through the gel. I already redid all buffers. Usually I used samples extracted from culture cells and now these are from tumor tissue. Anybody with the same problem?
I loaded 50ug of protein in a 10% acrilamida gel. Follow a photo of the membrane stained with ponceau.
Thanks!
Hi Yves,
In my opinion, you could try to load less protein (10 ug), it will give you better band resolution. Also your ladder is not ideal, so I would suggest to use a different gel and triple check your buffers :)
Hope it helps!
check your samples for any positively charging materials such salts. I have had this experience with DNA samples in agarose gels, so that a notable difference even between PCR products before and after purification (removing buffer and MgCl2) was seen.
Hi Yves,
In my opinion, you could try to load less protein (10 ug), it will give you better band resolution. Also your ladder is not ideal, so I would suggest to use a different gel and triple check your buffers :)
Hope it helps!
Hello,
you are having voltage problems. Voltage is not constant during your run so you experience such waved bands. This may rely on solutions (in the most case SDS or Tris-Gly) and sometimes on power supply.
Well, it appear that your proteins have runned well, in some samples your sample is less, but I can see that in all your samples you have protein. I think that some proteins (for example crystal proteins, viral proteins) are very resistant to denaturing. Try adding more SDS.
Hello Yves
It appears for two factors:
* There are a trouble with the preparation of gel
* There are different protein content. I think that you should be repeat the protein quantification
Best regards
I agree that your gel preparation is causing problems. Do you rinse the equipment in distilled/deionized water only? No soap? Including any glassware.
You could have a salt problem in the buffer. And/or your proteins are degraded.
I would suggest that you lyse your tumor cells for a longer period of time. It looks very similar to incomplete lysis of bacterial cells causing those same type of streaks, this is just an example as I understand that you are doing this to mammalian cells. Maybe your lysis of the tumor cells needs to be longer in length than your typical cell culture lysis. I mean I get that they are both just cells, but that would be my best guess if the other things suggested to you don't help your SDS-PAGE.
Hi
I think you might need to check the acrylamide stock that you are using. Also, the loading dye if not prepared properly poses this issue.
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