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Intern Needing Help

Western Blot Smear Vertical Banding SDS SDS-PAGE

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#1 doctorlambchops

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Posted 28 December 2011 - 09:26 AM

Hello Everyone.

I'm a research intern at MD Anderson Cancer Center looking for tips to make my blots look "right." My problem that I've been facing is that I keep getting these weird lines. I defrost and use WCL with inhibitors on application. I will also add that I accidentally boiled these samples longer than usual (99C for 20 minutes). I usually do 10 minutes.

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Edited by doctorlambchops, 28 December 2011 - 09:28 AM.


#2 doctorlambchops

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Posted 28 December 2011 - 10:06 AM

The protein of interest in my project is 98 kDa.

#3 bob1

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Posted 28 December 2011 - 07:28 PM

Check the salt concentration of your buffers, particularly the lysis buffer.

#4 mdfenko

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Posted 29 December 2011 - 02:40 PM

over boiling can cause protein loss due to aggregation. 5 minutes should be more than sufficient.

you can try ~65C for 10-20 minutes if you want to avoid aggregation during long incubations.

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