Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

Western Blot and Odyssey Image Software - (Mar/29/2009 )

Hi everyone,

I'm going crazy this is like the 7th that I have done this Western blot.
The first 2 times they worked but the last 5 times it just doesnt!
The problem is that we are using this scanner that uses fluorescent Ab as secondaries.
The last 3 Westerns that I ran I do not get any signal from IR 680 and its full of red dots
IR 800 however I can pick some signal from the protein though very faint. I wonder how can if my 2 Ab are going bad. I used a new dilution of my primary 1:1000 and for the secondaries 1:30,000. Any suggestions?
I am very troubled. ;)


Hi Bluelady,

Could someone else be viewing gels stained with Coomassie on this scanner, and not cleaning off the platform surface? I have heard that Coomassie can interfere with western blotting, although I'm not sure how. Coomassie also fluoresces at ~700 nm.

lab rat

-lab rat-