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Western Blot diffuse bands - (Oct/05/2015 )

Hi all, I have done Western Blot many times and this is the first time I have seen something like that on my blot! 

I ran whole cell lysates on 10% bis-tris gel and subsequently did a Western Transfer at 500mA for 1.5h. When I did Ponceau S after the transfer, the proteins on the blot had discrete bandings. I then probed with my primary antibody at 4oC overnight, and this is an antibody I have used numerous times so I doubt it is the antibody. The only thing I can think of might be that the Western transfer buffer has been used multiple times. 

I have attached an imaged. Would love to hear what you guys think!


Attached File


can you show us the ponceau stained membrane and how the bands match the blobs?


I don't have a picture of the ponceau stained membrane ;( 

Not sure if the bands match the blobs but they didn't appear like that on ponceau. Any idea about what the problem might be? 


blobs like those look like they're from a dot blot.


"bands" like those can appear if the protein ran out of the gel into a bubble against the plate then is reabsorbed but would also show when stained.


maybe some secondary antibody dripped onto the membrane just before you added the substrate?