Westerns from 3D collagen gels - (Sep/06/2007 )
I am trying to grow cells in varying concentrations of collagen. I would like to compare the activation of pathways like Rho/Rac under these different conditions but am unsure how to isolate cellular protein independent of the varying collagen concentrations which lead to very uneven loading on my Westerns. I tried using PBS with varying concentrations of acetic acid, but it does not seem to dissolve the gels very well (or at all, which doesn't make much sense...). For some application I may be able to use collagenase to extract the cells, but I suspect that will inherently change the activation status of some of the proteins I am interested in.
Help!! Any suggestions would be very much appreciated!
-ac8282-
QUOTE (ac8282 @ Sep 6 2007, 10:33 PM)
I am trying to grow cells in varying concentrations of collagen. I would like to compare the activation of pathways like Rho/Rac under these different conditions but am unsure how to isolate cellular protein independent of the varying collagen concentrations which lead to very uneven loading on my Westerns. I tried using PBS with varying concentrations of acetic acid, but it does not seem to dissolve the gels very well (or at all, which doesn't make much sense...). For some application I may be able to use collagenase to extract the cells, but I suspect that will inherently change the activation status of some of the proteins I am interested in.
Help!! Any suggestions would be very much appreciated!
Help!! Any suggestions would be very much appreciated!
Hi, three possibilities:if it's a GEF assay you want to do, don't you have to perform an IP?
Second: extract protein load same amount in ul run the WB, check b actin and adjust the volume of your samples on the basis of that...rerun the wb..long but cheap
Third: people in my lab used BD cell recovery solution( cat n 354253)..it's expensive but seems to work fine...good luck
aleric4u
-aleric4u-