Good loading control for native gels? - (Oct/21/2012 )
I have recently found that one of the proteins that I am looking for in my homogenized mouse brain samples (both in plain PBS and also in RIPA lysis buffer) can only reliably be detected when the sample is run under purely native conditions (native gel, no heating, no reducing, no SDS in loading or running buffer or gel). Western blotting for the protein under these conditions produces beautiful results with little background and nonspecific labeling. However, I'm having one heck of a time finding a loading control that works for native gel westerns. Beta tubulin (Ab6046 from Abcam) appears to dimerize under native conditions and no longer has a band around 55 kDa and both GAPDH (Ab9485 Abcam) and alph actin (Sigma whole antiserun a2668) are not visible when I probe for them. I am wondering if anyone knows of a loading control that works for Western blotting under native gel conditions? I am considering ordering an anti-beta actin Ab that claims to recognize both the native and denatured form. Does anyone have any experience with this? This problem has caused much frustration and any help would be greatly appreciated!!
try BSA, ovalbumine, apoferritin, alcohol dehydrogenase, thyroglobuline