Hello,I am a PhD student at the University of Siena. I am trying to detect the levels of Glut 4 protein in a western blot on some skeletal muscle tissues lysates isolated from mice. I am running into problems because I can't see it in a 4-12%Precast gel whereas in a 10% homemade gel I can. There is a specific reason? Moreover, the identification of Glut4 protein in 10% home made gel is not constant. Is it a problem about the sample preparation? There is a specific procedure to lysate the tissue to allow a better Glut4 solubilization?Thank you in advance for the help!
Luisa: Your problem of inconsistent detection of Glut4 band on WB in home-made PAGE, and no band on commercial precast gel, may point to the issue of the antibody used rather than different gels. Based on our experience, you may want to conjugate your antibody directly to HRP to avoid any non-specific bands around 40-60 kDa which may overshadow your real band by the traditional secondary anti-IgG-HRP.
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