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problem with SDS-PAGE


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#1 proteins

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Posted 02 June 2009 - 06:21 AM

Hello everybody,

I am running SDS-PAGE gels with my protein of interest. The molecular weight of my protein is 190 kDa. I am trying to inhibit the protein and then see if it gets phosphorylated with another kinase. The molecular weight of the inhibitor protein is 568 g/mol. The mechanism of action of inhibition is that it binds to the protein and competitively prevents activation by an activator.

I add the protein, inhibitor and kinase in to an appendorf tube, then add DTT and SDS-sample buffer after incubation, and heat it for 5 min at 80 C. I have done this almost 7-8 times until now. Almost every time, the samples get stuck in the gel and doesn't seem to pass throught the gel. I used mercaptoethanol in SDS-sample buffer before, but now i use DTT. I changed all the buffers and prepared them again and set the pH of running and stacking gel buffers to 6.8. I wash the top of the running gel very well before adding the stacking gel for polymerisation. After having done all of this, i am still not able to make the proteins pass through the gel. I dissolved the inhibitor in DMSO. Could that contribute to anything like this when heated? Or is there any other reason? PLease help me. I am getting frustrated.

Or do any of you know any other methods to check if a protein is phosphorylated by a kinase.

#2 madrius1

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Posted 02 June 2009 - 06:48 AM

Have you tried switching back to BME?
Also, you may want to check every constituent of you mix individually, to figure out what impairs your protein to get in the gel.

If you put other mix of protein, like a cellular extract, does it enter the gel? Or is it a problem specific to this assay?

#3 proteins

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Posted 02 June 2009 - 11:52 AM

Have you tried switching back to BME?
Also, you may want to check every constituent of you mix individually, to figure out what impairs your protein to get in the gel.

If you put other mix of protein, like a cellular extract, does it enter the gel? Or is it a problem specific to this assay?


I encountered this problem while i was using BME with this assay and another assay. So, i changed to DTT. Now the other assay is working fine, but this assay still doesnt. I have tried with partially purified cell extracts and it worked well. I tried individually and everything works. It is only when i mix all of them and try to load on the gel, everything is going wrong.

Any other suggestions please?

#4 bob1

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Posted 02 June 2009 - 04:45 PM

Are you using the right percent gel?

#5 proteins

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Posted 03 June 2009 - 01:34 AM

Are you using the right percent gel?


The protein is a GST-fusion protein which has only a partial sequence and therefore, it should show a band at around 79 kDa. I used a 12.5% gel before and i also used a 10% gel recently.

Do you think i must change to some other % gel or change the type of gel from SDS-PAGE to any other. I am not familiar with other types of gels (like Bis-Tris PAGE) and under which conditions they are used. But for this situation, do you think changing the gel would show any effect? Or how about native gels?

#6 bob1

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Posted 03 June 2009 - 04:19 PM

Just checking the obvious, it could have been that you were looking for a 400 kDa protein on a 15% gel. Your 10% should be fine.




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