Western Blot diffuse bands (View forum version)



tofuj

Posted 05 October 2015 - 08:46 PM

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!

 

 sad blot.bmp   4.5MB   612


mdfenko

Posted 06 October 2015 - 04:17 AM

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


tofuj

Posted 06 October 2015 - 02:09 PM

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? 


mdfenko

Posted 07 October 2015 - 04:14 AM

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?