The protein of my interest is of 69 k Da, but i observe a nonspecific band at 55 Kda along with the specific band, even after increasing blocking i see this band, it also stains ladder at same position (55 KDa). Antibody data sheet also show this band, but i don't understand what could be the reason. Is their any way to remove this. I use PVDF membrane.
What kind of antibody is your primary (which species) ab? And how do you detect it? Given the weight of 55kDA I would guess that you are detecting the heavy immunoglobulin chain of your primary ab.
Dear Christian, my primary antibody is raised in Rabbit and anti rabbit HRP secondary was used followed by detection by ECL prime kit
Dear Priynaka,
Presence of your unwanted band in the marker line suggest the gel-running apparatus is dirty... Could you show the picture of your blot to verify this hipothesis?
Best - jan
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