Problems with Western blotting of tissue homogenates - (Feb/29/2008 )
Hi everyone,
i urgently need some help here...
I'm doing western blotting of mouse brain tissue homogenates, extracted in RIPA buffer. From the western blots I'm trying to quantitate the differences in a specific protein (using GAPDH as a loading control) in different mouse brain lysates. The blots I get are clean, however quantitation always yields variable results. I've tried running the same set of samples on 2 gels and performing the western blot at the same time, so that i get results on the same film to eliminate any differences in exposure during autoradiography. However there's still the problem of inconsistent results, with as much as 100% variation in quantitated protein levels for the same samples, on the same film. Does anyone have a problem with using tissue lysates during western blotting? Please let me know if you have any suggestions for troubleshooting this...
Any advice will be greatly appreciated, thanks heaps in advance!
madpie
i urgently need some help here...
I'm doing western blotting of mouse brain tissue homogenates, extracted in RIPA buffer. From the western blots I'm trying to quantitate the differences in a specific protein (using GAPDH as a loading control) in different mouse brain lysates. The blots I get are clean, however quantitation always yields variable results. I've tried running the same set of samples on 2 gels and performing the western blot at the same time, so that i get results on the same film to eliminate any differences in exposure during autoradiography. However there's still the problem of inconsistent results, with as much as 100% variation in quantitated protein levels for the same samples, on the same film. Does anyone have a problem with using tissue lysates during western blotting? Please let me know if you have any suggestions for troubleshooting this...
Any advice will be greatly appreciated, thanks heaps in advance!
madpie
we have had some problems trying to quantify the western blots. The signal between samples are linear in a narrow range. so unless you have loaded samples in that range, its difficult to quantify. It can be done but tricky.
Hi Scolix! Thanks for your reply! 
I'm currently loading apporox 20ug per lane. Was just wondering what amount you loaded when it fell within the linear range? What sort of tissue lysates were u using too?
Cheers,
madpie
We do not quantify anything in western. We load equal amounts of a sample (and also different dilutions) and control and if there is too much signal, then we reduce the load volume and then it might indicate better linearity. What we actually do is to get an impression if one sample has more or less of a particular protein than the control. This might sound not right to some people , but this is how my PI likes us to do Westerns.