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Looking for new endogenous control for WB


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#1 Sumpf

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Posted 27 April 2009 - 02:32 AM

Hello,

I had problems with stripping my mebrane so I decided to cut it instead. But now I have a problem with the endogenous control. One of my protein of intrest is just in the same range as beta-acin. So what could I use instead. I thought of Tubulin but this is also running in the same range of a protein of intrest. Ist there somehing smaller than 30 kDa or larger than 60 kDa i could use?

Thank you for your attention.

#2 yobou

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Posted 27 April 2009 - 04:49 AM

unfortunately the 3 main loading controls (GAPDH, beta actin, and tubulin) are in the range of 36, 43 and 55 kDa. in some papers they use a non specific band appearing on the blot as a loading control. but stripping if done properly should not be a problem at all for loading control.

Edited by yobou, 27 April 2009 - 04:50 AM.


#3 Dr Teeth

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Posted 27 April 2009 - 05:58 AM

unfortunately the 3 main loading controls (GAPDH, beta actin, and tubulin) are in the range of 36, 43 and 55 kDa. in some papers they use a non specific band appearing on the blot as a loading control. but stripping if done properly should not be a problem at all for loading control.


Use RNA pol II (~220 kD).

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#4 Sumpf

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Posted 27 April 2009 - 06:13 AM

unfortunately the 3 main loading controls (GAPDH, beta actin, and tubulin) are in the range of 36, 43 and 55 kDa. in some papers they use a non specific band appearing on the blot as a loading control. but stripping if done properly should not be a problem at all for loading control.


Stripping is not working. I use the same protocol and the same solutions as the others do in my lab but somehow my proteins are always gone. I transfered longer, used different membranes, but it's as it does not like me.
Even when I am doing it with together with someone else, they disapear.

Use RNA pol II (~220 kD)


is this published somewhere? And do you know also something for the cytoplasmic fraction?

Edited by Sumpf, 27 April 2009 - 06:15 AM.





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