I want to detect GLUT4 translocation into the plasma membrane of L6 rat skeletal muscle cells upon sample treatment. As a test run, I extracted the sample treated cells with lysis buffer (0.1% NP-40) and fractionated into Plasma Membrane (PM), post plasma membrane (PPM) and cell lysate. Then proteins from each fraction (25 micrograms protein per lane) was separated by 10% SDS electrophoresis, trasfered to PVDF membrane,treated with primary antibody (anti-GLUT4, 1:500) followed by compatible secondary antibody and subjected to cheminofluorescence detection. However, the signals obtained even for insulin were weak, whereas stronger bands were observed in all the cell lysates. Therefore, how can I optimized the GLUT4 band strength in plsma membrane fractions?
Hi,
I think the problem is not the optimization of antibody but the extraction of membrane fraction per se. Isolation of membrane fractions is sometimes tricky. Try using RIPA buffer since it is more robust in isolation of membrane fractions.
However, immunocytochemistry and z-stacking may better help to be sure there is translocation to the membrane.
Hope this helps.
Santosh
Dear Santhosh,
Thanks a lot for your comments. Actually, I use 2 X RIPA buffer to extract cell lysate which I can observe very clear GLUT4 bands on the blot. Our laboratory is adapting the rapid preparation method described by Nishiumi and Ashida, 2007 which use 0.1% NP-40 containing buffer to prepare plasma membrane. I`ll check the extraction with RIPA buffer. Kind regards.
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