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bands of protein seems like it did not move


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#1 zienpiggie

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Posted 26 April 2010 - 09:33 PM

Hi all,

I'm pretty new at western blotting and I am trying to detect this protein with a size of approx 60 kDa, but when ubiquitinated it will be approx. 100 kDa. I ran my first blot the other day but did not find any bands at either 60 kDa or 100 kDa, but more all the way up there to a point that I concluded that my protein most likely did not travel across the gel. It was 10% running gel for a 60-100 kDa so I thought the gel concentration should be fine. The actin band for example was right where I expected it to be. I then thought it was due to dimerization as the protein naturally is associated with other protein in cytoplasm, so then I thought may be I haven't denatured it enough. It was boiled for 2 minutes prior to loading. I am just wondering if anyone has experience with such observation?

Thank you.

#2 mdfenko

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Posted 27 April 2010 - 07:13 AM

you could try heating the sample at 65C for 10-20 minutes instead of boiling. sometimes boiling will cause the protein to aggregate, even in the presence of sds and reducing agent.
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#3 zienpiggie

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Posted 29 April 2010 - 04:14 PM

Thanks mdfenko. I'll give it a try.




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