I would really appreciate some help with this.
I am studying a membrane protein, around 40KDa but I am unable to detect the majority of the protein by SDS-PAGE as it seems to stay stuck at the top of the gel.
I have tried whole cell lysates (2x SDS+5%2-ME), NP-40 lysis buffer, RIPA buffer and dedicated membrane protein extraction kit but most of the protein refuses to migrate through the gel. I am over-expressing the protein by transfection and have tried both lipofectamine and Ca/P methods but these don't seem to be the cause.
I have tried NuPage 10% Bis-Tris gel and Criterion TGX AnyKD gel. Both show the same thing (example attached).
I tend to run the gels at low voltage (around 100V) for 1-1.5h in general.
I have not tried using DTT yet.
Any ideas would be really appreciated.