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EMSA supershift - increased band intensity?


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#1 kbee

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Posted 08 May 2009 - 10:00 AM

Hello you EMSA experts,
I've successfully performed several radioactive gel shifts with short oligonucleotides and different nuclear extracts using Promega's gel shift assay system . I've recently started supershift experiments with Abs to confirm the identity of the bound protein. My problem is that instead of shifting the band up in the gel, addition of the Ab leads to an increase in the density of the original band. This is reproducible and the increased intensity correlates with the amount of Ab added (1-2ug). This isn't due to the buffers present in the Ab, as my IgG control has no effect. Also, I only see this with my DNA probe of interest, when I use a labelled consensus probe in the supershift (with identical reaction conditions) I do see a nice upwards supershift. Any explanations?

#2 drjcroberts

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Posted 12 June 2009 - 11:04 AM

hi,

this is a very common thing with supershifts

what happens is that the incubation with anibody causes odd things to happen:
1) increased solubility of the main 'shifting' protien in solution. its then more able to bind to the DNA. this can also be caused by the anibody (i.e. VS IgG) due to different purification technolgies that the company uses to make it.
solution: dilute anibody in 2xbinding buffer before use. make a wider range of dilutions.

2) conformational change in the protein and disaoosication. remember, this assay is an affinity SYSTEM. there is affinity and disassocisation- its dynamic. what can happen is that the anibody is effectively making the protein more stable but once the DNA-protien complex forms the anibody disaoosicates on a gradient. thats the theory anyways! essentially the DNA-protein-antibody is unstable- even if your + control works, this can happen.
solution: add sterile filtered BSA to the reaction at 0.1%

3) the anibody 'mops up' non-specific proteins, some of them isofoms of the same iteracting protein. this is especially common if your shift is large MW
solution: try using purified proteins or a ChIP-grade /different antibody

4) the shift you see is a complex with another protein. do you see a reduction in a lower band in those lanes?
solution: try a range of different competitors and start over... yikes! of course this could be significant... remember you cant tell what is what even if you have a sfhift of equal size it could be conicindental!

hope this helps, references i have if you need

J




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