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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Western blot funneling
Posted by: Laazie83 (IP Hidden, New member, 2)
Date: December 8, 2005 02:00PM
Hi,
I'm using trying to get rat white fat samples to run properly on my gel. 120V, max amps is what I'm using now. My problem is that every second loaded sample is running into a funnel...but not all of them. Should I drop the voltage? Do you think there is protein aggregation (if so, what would you recommend?) My protein's around 31kDa. I started from scratch with the extractions, thinking something had happened there, but now these newly extracted samples are funneling more often than the last ones! (Still not every one though). Any ideas? By the way, I don't pour my own gels. I use the NuPage system. All the samples have undergone the same preparation so that discounts any local interference of different solvents and such. Edited 2 times. Last edit at 12/08/05 05:55PM by Laazie83.
Re: Western blot funneling
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: December 12, 2005 06:47AM
The electrophoresis funneling can be caused by over reducing of protein samples. Try use DTT and 2-ME as reducing agent in your lysis buffer. Hope it make sense.
Re: Western blot funneling
Posted by: Laazie83 (IP Hidden, New member, 2)
Date: December 13, 2005 02:54PM
HI. Thanks for the advice. I had used DTT already, but perhaps I hadn't allowed it to dissolve sufficiently. I'll try include the 2-ME also.
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