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Puzzeling western blot results, missing band

western blot missing band

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#1 everyday lab rat

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Posted 17 May 2012 - 03:03 PM

Hi All,

I am currently working on a pancreatic patient xenograft model. I receive either fine needle biopsy samples or solid tumor mets for transplant into immune compromised mice. We are having quite a bit of success.

I recently did a few quick western blots on some lysate that I made from 2 patient primary tumors and the resulting tumors that grew in the mice. I use ACTIN as a control to make sure that the lysate prep is good, p-ERK is one of the proteins we are interested in so I ran that blot at the same time. As you can tell from the image, Pt. #2's primary tumor does not have a band for ACTIN. I repeated the blot to confirm that my prep for the blot was good. This same sample has a really strong band for p-ERK which to me, shows that the lysate sample is good.

Does anybody have any idea why there would be no ACTIN band for this sample?

Thanks a bunch!
Dawn

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#2 almost a doctor

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Posted 18 May 2012 - 12:51 AM

Could you give us some details on how you perform your WB? The more detail the better we can help. That p-ERK band on the sample missing Actin, looks a lot like the other ACTIN bands (but upside down), which is kind of weird, and as I don't know what you've done is hard to tell whether this could actually happen. The missing ACTIN band lane looks too clear, almost as if there was no sample there.

Are the results from 2 different gels/blots, or is the same membrane stripped or cut?

#3 everyday lab rat

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Posted 18 May 2012 - 03:14 PM

Those are separate blots that I ran at the same time. The ACTIN blot was a repeat (I had the same exact results for the first ACTIN blot). For the repeat ACTIN blot, I prepped a new sample from a different aliquot of lysate to rule out error while prepping the sample for the first blot. When I prep samples to run a gel, I make up enough for 6 or 8 gels so I can just aliquot out and freeze the extra. These two blots were run with the freshly preparred sample.

I usually run the gel @ 50V until the blue band is halfway down the gel, then I up it to 100V to finish off. Transfer was standard, 100V for 1hr, cold. The standards transferred beautifully. The membranes are blocked in milk buffer with tween for an hour. To make life easy, I incubate the 1' antibody O/N, @4'C on a shaker. The ACTIN antibody is from Cell Signaling, it's conjugated with HRP so no 2' is needed. In the morning I do 4 washes in 1xTBS/T (20mls/10min). I've been using the Pico chemiluminescent kit to develop my blots and xray film. The p-ERK needed a 2'ab, so that blot was developed an hour later than the ACTIN blot.

This just really puzzles me. I've never not had a band for ACTIN. I've had cruddy bands, but never one that was completely missing. I wasn't sure if perhaps there was something in the lysate that may interfer with the antibody. Lysate from human tumors can be cloudy and sometimes have a film layer on top after I spin the sample down.

#4 everyday lab rat

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Posted 24 May 2012 - 03:25 AM

Bueller? Bueller? No ideas from the collective?





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