I have a problem detecting the small protein of 7kb in western blot. Hers is the procedure that i have been using
my protei has two isoforms of 37KD and 7KD with the flag tag. some other people have already show i different system and we believe it occurs in two isoforms in our system too looking at the protein sequence.
i have been using biiorad precast tris glycine gel gradient of 4-20%. run at 180V for 45 mins. do semidry trasfer at 10V for 1 hr in 0.2micronm PVDF. i use PBS buffer and wash in PBST. the marker in the membrane are quite visible but when i do imaging, the 37kD protein only gets detected. somewhere in the forum, i saw heating the membrane in boiling water to fix the small mw protein, i tried that too but i could see nothing at 7kb band are.
What is the suggestion for doing western blot for small MW protein like 7kb?