Hello everyone,
I have a colleague that is running a gel electrophoresis and immunoblotting phospholamban, p-phospholamban-Thr17 and p-phospholamban-Ser16 in rat cardiac muscle. She is loading 5 and 10 micrograms of total protein, using 0.18M DTT (fresh), 1xLDS and heating her samples for 30min at 95C.
She aims to dissociate the pentamer into monomer and so, quantify relative amounts of PLB and p-PLBs.
However she can still detect the pentamer with her Ser16 antibody, but not with PLB or Thr 17.
Does anyone have experience with this technique or would like to share your thoughts? That would be very appreciated.
Many thanks.
Hi Aline, are you sure your PLB and Thr 17 antibodies recognize the denatured antigen. This can be an issue with some antibodies. For denaturing the samples use 5% beta mercaptoethanolamine to the samples and heat in a glass tube for 5-10 min. As a control lane also run your sample without a reducing agent and heating.
Good luck, let me know if that helps or not.
Hello Bernard. I do appreciate your attention.
Thanks for your suggestion, I will discuss this technique with my colleague. I also told her that she can check the antibody features with the technical support of the company she is buying it from. I don't believe she tried the protocol you have suggested here. Again, many thanks for your help!
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