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Protein solubilization and Western Blotting


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#1 tltan

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Posted 09 December 2004 - 09:55 PM

Hi guys,

I am currently working with viral proteins expression and western blotting. However, I havent been successful.

The viral proteins were cloned into pCDNA3.1+ or pXJ vectors and transfected transiently into cell lines. After that, the cells were pelleted down and solubilized using sds sample buffer. Sonication was carried out followed by SDS-PAGE.

Antibodies were purchased commercially and havent worked since. doing control experiments using commercial actin reveals western blot protocol is working. Am wondering what is wrong with my whole step.

thanks,
tltan

#2 tltan

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Posted 09 December 2004 - 10:04 PM

Hi,

Would like to clarify another problem regarding SDS-page in this same thread. Sorry to bother you guys!

I have some total cell lysed poteins solubilized using 7M urea/ 2M thiourea, and proteinase inhibitor for 2D electrophoresis. Can I run the same samples onto SDS-PAGE? Do i need to do any equilibration step or whatsoever for the proteins to 'run' in smoothly?

Thanks in advance for any help rendered.

Cheers,
tltan




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