I'm doing westerns with an anti ubiquitin Ab on total cell extract.
Attached is a picture of my western blot.
As I expect ubi to conjugate to proteins of all sizes I expect to get bands of all sizes. And I do, in a way. However, all big proteins come out as negative bands and I do not know why. Anybody ever seen this or have any idea on whats going on?
I load 2micrograms of total protein on a 4-20% gradient gel and dilute both 1st and 2nd Ab 1:3500. I have low signal; the blot shown is exposed to a film for 15 minutes.
all input would be highly appreciated.