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Dot instead of a band on precast gel (running funny!)

Dot instead of a band on precast gel (running funny!) - Western Blot Forum

Dot instead of a band on precast gel (running funny!) - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 03-18-2009, 07:55 PM
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Unhappy Dot instead of a band on precast gel (running funny!)



I have a preculiar problem with samples running weird on my gel...precast Invitrogen gels (gradient 4-12%, 12-15 wells). My samples are protein derived from tissue or cells. I use Mops buffer for running. While running the samples I notice that instead of horizontal line there is a wave and some lanes have formed a mountain-like structure and are not running right. At the end of the blotting, post-antibody staining, these lanes look like a dot instead of a band at expected size. I don't know what is happening and it is always different samples. Tried changing a lot of stuff but I wish I knew what this was because of. I am not the only one in lab who is having this issue and we've been through numerous gels and samples because of this mysterious problem.


Please help! PLEASE!
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Old 03-18-2009, 09:33 PM
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Default Re: Dot instead of a band on precast gel (running funny!)

It's likely the buffer or protein concentration is too high.
Can you change out of MOPS?
I never use it, Tris-HCl gels and TGS buffer, works for every application I've encountered so far.
Are your gels Bis-tris and specifically for MOPS? did you also get the MOPS from Invitrogen (like --> [Only registered users see links. ] if not does it contain SDS?

Lastly, make sure the gel isn't expired.
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Old 08-05-2009, 09:11 PM
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Default Re: Dot instead of a band on precast gel (running funny!)

I just had this effect on a gel today on a 0.8% agarose gel in TAE w/EtBr the first ladder looks fine but as you go across the gel the bands begin peaking to look almost like the Atari logo.
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Old 10-15-2009, 09:24 PM
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Default Re: Dot instead of a band on precast gel (running funny!)

I am having this same problem as well. I also use the invitrogen precast gels (brand new) and invitrogen MOPS running buffer. I am certain it is not a buffer problem because my protein ladder always runs fine. It has to be the sample for some reason. I load my gels at about 40ug protein per well (I know this is on the high end but it shouldn't be an issue). This problem has been haunting me for a while now and I can't get rid of it. It seems to happen every other time I run a gel. I have tried adjusting my cell lysis conditions (more/less stringent wash, more/less sonication, more/less spin steps) but nothing is fixing the problem. I also have had this problem with homemade RIPA buffer, thermo-brand RIPA buffer as well as another lysis buffer recipe that I found. I am out of ideas...
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