5 replies to this topic

[LabGirl]

We are having issues with our ChemiDoc for imaging Westerns and wondering if anyone has suggestions? The Western blot conditions have been optimized and we were getting consistent results previously. However, now we are getting grainy/spotted images using our ChemiDoc. It is happening to multiple users with multiple blots. It's only happening when we image Westerns; our agarose gels are not grainy, so we don't think its the camera or dust. We are using Immun-Star Western C Chemi Kit from biorad at 1000 ul total on each membrane. When the ChemiDoc is imaging, the pictures are black and no bands are seen, however when we go to review the image, we just see a grainy picture. An example image is attached.

Any suggestions would be helpful, thanks!!

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[mdfenko]

i can think of a couple of possibilities:

the camera resolution is set wrong

the vessel in which you are processing the blot needs to be thoroughly cleaned

Edited by mdfenko, 07 June 2012 - 11:55 AM.

[LabGirl]

Hi mdfenko,

Thanks for the input. I am sure the camera resolution is fine-sometimes the Western blots do work (and work well), and the camera itself should be fine: we image agarose gels and other items on this ChemiDoc with no problems. We have been consistently cleaning very thoroughly the entire time, and at every step of the blot, but are still having the same problems. Any other options you can think of? I think it could simply be an antibody problem, but sometimes this antibody will work, sometimes not.

[mdfenko]

what do you use to block?

maybe your block is sometimes not fully solubilized or has precipitated.

Edited by mdfenko, 15 June 2012 - 08:34 AM.

[LabGirl]

Hi mdfenko,

I thought the same thing, however we now know what was going on. I was posting this for my grad student, and apparently she had reduced the amount of the protein sample she was loading by more than 50% and failed to mention it, so I think that was the issue. I believe the grainy appearance was the imager trying to pick up a signal where there wasn't any. I suggested increasing the amount of protein she was adding and also using a positive control.

Thanks for the reply!

[science noob]

What is the exposure time for this image?