I have been trying to run Western blots for ALIX and CD63 using SBI's antibodies, but I get big, bright bands at ~35 kD for all of my samples. The samples were isolated using SBI's ExoQuick ULTRA EV Isolation Kit for Serum and Plasma. I followed the Exosome Antibodies User Manual’s protocol for blotting, the only modification made being loading ~ 25 µg of protein into each well instead of 20 µg. I don’t know of any serum or plasma protein contaminants that size that could account for the bands. Any guesses for possible contaminants?
Sure (rather 37 kD), but I do not think that he wanted to see GAPDH using ALIX and CD63 antibodies.
However. if this is not the correct band for your proteins of interest, I would say that your antibodies are not specific for your targets (for ref. see: Baker, M. NATURE. VOL 521, MAY 2015).
hi
It is related to the secondary antibody reaction with the immunoglobulin present in the loaded sample (37 kd band). To ensure this, you can re-probe your blot paper and then expose it to the secondary antibody only.
good luck
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