I'm using HEK cells stably expressing a construct and I am examining the cells using sequential fractionation. My fractions are based on the following extraction steps:
a) Total lysate
b) Freely diffusible protein: Hypotonic lysis
c) Membrane bound protein: 1% Triton x-100
d) Detergent insoluble protein: 8M Urea + 2% SDS
I'm hoping to use GAPDH in the total lysate as a loading control but I'm wondering if I should expect to see any GAPDH in the detergent insoluble fraction? I've seen some papers on pubmed referring to insoluble GAPDH and I'm wondering if anyone has any experiences they can share? Thanks!
GAPDH is everywhere!
When I do cell fractionation I always observe GAPDH in mitochondrial, cytoplasmic and even nuclear fraction. With ultracentrifugation at 240 000g GAPDH is found in the supernatant and the ribosomal pellet. I loose GAPDH in the ribosomal pellet under sucrose cushion with 500 mM KCl but I also loose a lot of protein of interest for my studies.
GAPDH binds RNA so make a RNAse treatment if that do not interfere with your experiments.
Good luck,
Cheers
GAPDH is the worst marker ever. Use cyclophilline A to show that fractions are free of soluble proteins or vice versa.
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