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Help with western blot!!


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#1 lilin_hust

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Posted 04 April 2014 - 03:23 PM

I did WB with the same sample, the first time the band is very good, and signal is strong, however, when I take out the sample from -20 degree, and boiled again for another 10 min (when I first extract the protein, I also boiled 10 min), and I use just same primary antibody and same concentration, then I didn't get any band.

 

Why is this?? anyone has any ideas? I think it is probably the proteins are degraded when I did the second 10 min boiling. Has anyone also has this problem??

 

Thanks in advance!!

 



#2 bob1

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Posted 04 April 2014 - 11:55 PM

Proteins can aggregate and precipitate out of solution with prolonged boiling.  If you have boiled them once, you don't usually need to boil them again.






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