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Expression of Proteins for western blot


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#1 tltan

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Posted 07 September 2004 - 07:55 AM

:D
Hi dear forumers,

Just like to check if anyone has any experience regarding expression of proteins for western blot analysis using cloned plasmids.

I have cloned a gene coding for a specific protein into an expression vector pCDNA3.1+, and would like to express the proteins and run it on a sds page.

As such, i have transfected the cloned vector into cell line and extracted total proteins. With that, I ran a sds page. However, western blot did not yield any bands upon exposure.

2 questions,
(1) do you guys heat up the proteins before loading it onto the SDS-PAGE gel?
(2) Is there anything i missed out above which could have resulted in the "no band" case.

Thanks a lot in advance for any inputs here.

tltan

#2 Sprag

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Posted 07 September 2004 - 09:43 AM

Yes, always boil your samples for 5-10min before loading (to denature the protein!!).

Is your antibody working?! Can you detect endogenous protein? or are you using tagged protein? If so, then try to express another protein with similar tag and use that in your western as positive control.

Always try to include positive control so you know your technique is working (a general advice!).




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