I have been running western blots to verify the presence of a protein in my co-IP but a strange rectangle keeps showing up on my membrane and blocks the signal when I image it. I reran my western blot, but the rectangle reappeared again. Does anyone know what it could be? Any suggestions would be appreciated, thank you!
Info: I used a PVDF membrane and incubated it in methanol for 2 min. I have successfully detected bands for my co-IP before. I know the problem is not with the imaging system because my colleague detected her membrane without the rectangle earlier in the day. I have increased the contrast all the way up so the rectangle is visible. Only my input band is visible on the very left.
Hi,
this rather looks like a defect to the membrane of an issue during activation. Could it be that something this shape was pressing on the membranes during storage or so? I have seen something like this regularly due to touching of the membrane to harsh with forceps (however the area is much smaller in such a case)
Sven
If you image exactly the same as your colleague (same imaging modality, technique, and detection method), then this looks like an issue of transfer. Have you checked your transfer cassette? Sometimes the sponges, especially in older labs, can get a bit crusty and that buildup impacts everything else. If you do dry/semi-dry, it could be uneven pressure during rollout of the transfer stack.
If you perform imaging differently (different film holder, wetting the stage in a CCD detector, not using a plastic sheet, etc) then your culprit is drying during imaging.
I'd lean on imaging issues because you have some very well-patterned noise, which makes me think there's a problem with the detector that builds up over time. Perhaps cooling issues? Unless that's an artifact of compression/this site in presenting images.
hi
It looks like the ECL droplet has not dried . You can wash the paper in the TBS-T buffer and Repeat the imaging with a weaker ECL substrate .
good luck
Sven Reister Hi Sven, that is a possibility. My colleague thought it might have been the glue on the outside of the membrane to keep the roll of PVDF together. Thanks for the suggestion!
Colin Germer We wash our sponges well after using it but that's a possibility. I will double check the next time I transfer. Can you elaborate more about the cooling issues? Thank you!
Reza Hadavi Thank you!
The cooling issue can arise in CCD imagers. Basically, the detector has to cool to a certain temperature, usually somewhere around -15C to minimize noise due to small defects in the sensor. This noise doesn't manifest itself in short exposures, but longer ones (>2 seconds) will have it.
I say this because that square you have has some very neat lines on it as well as throughout the image. Though I doubt that the square itself is caused by any cooling issue.
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