We've ran many blots using this protocol and have never seen contamination like this. I will try soaking all dishes and sponges in 70% ethanol. Any other thoughts?
Thanks for taking a look!
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Protocol details:
Used BioRad precast 4-20% gels
Protein samples prepared with 2x Laemmli Sample Buffer containing bromophenol blue and 2-mercaptoethanol
Ran gels at 90V until loading dye had run off gel (gels ran in same chamber)
Transferred to nitrocellulose membranes at 350mA for 1 hour (blots transferred in same chamber, separate sandwiches)
Blocked in Odyssey blocking solution for 1 hour
Incubated in primary antibodies overnight, washed three times in 1x Tris Buffered Saline (TBS) + Tween, incubated in secondary antibodies for 30 minutes, washed three times in 1x TBS+Tween, final wash in 1xTBS
Imaged on LiCor
I agree with Hans, a Field Application Specialist from LI-COR also made us aware of coomasse contamination. In addition, bromophenol blue might be the cause.
Best
Thomas
I agree with both Hans Bakker and Thomas Gantert. Anything blue can show up in the red channel and with the spread and brightness commassie seems the likely culprit, but I have also seen problems with Sample buffers with blue dyes (I make mine with orange g now). Another potential problem that can cause background is bacteria contamination in the blocking buffers/antibody dilutions, or dirty boxes, although I don't think that is usually as bright. Also, since you mentioned dirty sponges, they tend to have a more speckled effect.
You should also check out the document licor put together called "whe good westerns go bad" (I attached the link). It's very helpful and has lots of pictures for examples of potential problems.
https://m.boxenterprise.net/shared_item/https%3A%2F%2Flicor.boxenterprise.net%2Fs%2Fdq6jb8sgnkiwlas0g99b5hq0tsuvcyyz
we also had a similar kind of contamination and our Licor contact person told us that the most common reason for that is a contamination with BPB sample buffer. They suggest not to let the blue front running out of the gel (because when it's in the buffer, it's contaminating the chamber), and then to cut if off your gel before blotting.
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