3 replies to this topic

[Sumpf]

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.

[yobou]

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.

[Dr Teeth]

yobou, on Apr 27 2009, 08:49 AM, said:

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).

[Sumpf]

yobou, on Apr 27 2009, 02:49 PM, said:

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.

Quote

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.