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high MW bands are white low MW bands are black?!?

high MW bands are white low MW bands are black?!? - Western Blot Forum

high MW bands are white low MW bands are black?!? - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 11-25-2010, 10:25 AM
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Question high MW bands are white low MW bands are black?!?



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.
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File Type: jpg ubi western 251110_whitebands.jpg (94.6 KB, 11 views)
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