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Cold probe doesnt compete away bands


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#1 Mortis

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Posted 27 May 2010 - 08:18 PM

Hi

Recently ive tried doing non-radioactive EMSAs with an agarose gel protocl, and it seems to work ok, but i have one major problem: my cold probe does not compete away my observed shift. I thought i might just be seeing a non-specific band, however when i load a pure sample of the transcription factor protein (i.e. pure recombinant human Glucocorticoid Receptor) which should bind my probe (i.e. a well described Glucocorticoid Receptor binding sequence, otherwise known as a "GRE") instead of the nuclear extract i see the same thing - no competition from the cold probe. This has happened for every GRE probe ive tried. Oddly, the free probe band intensity often decreases noticeably in my competition lanes.

Ive made my probes both by buying biotin-labeled oligos (from Sigma) and also manually labeling oligos using the Pierce biotin-labeling kit. I add roughly equal molar amounts of sense and antisense together, mix, then use a PCR machine to heat to 95 degrees, then cool by 1 degree per cycle - for 70 cycles.

Obviously the cold probe is far more concentrated than the labeled probe, but is that possibly affecting the annealing so that i dont have a functional cold probe? Does anyone have any other ideas as to what is going on?

Thanks in advance

#2 HomeBrew

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Posted 28 May 2010 - 03:41 AM

I don't have experience with EMSAs, but maybe it's a detection sensitivity problem? If your cold probe displaces 99% of your labeled probe, would 1% label still show up? Maybe you're asking too much expecting the band to be completely off -- the cold and labeled probe exist in equilibrium, there will always be some label, right? Especially in light of "oddly, the free probe band intensity often decreases noticeably in my competition lanes"...

How are you detecting your signal? If it's qualitative, like chemiluminescence, maybe you need a more quantitative method?

#3 Mortis

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Posted 14 July 2010 - 02:46 PM

Hi again - sorry i took so long to reply. I thought it would give me an email warning if someone replied to my thread, but it didnt (ll have to check my settings on that)

Anyway, yeah i had thought that possibly the sensitivity of the hot probe is not giving me proportional signal. However, i DO see proportionally less signal with reduced nuclear protein added - its just that my cold probe competetion band for each particular amount of protein shows the same intensity as the hot probe alone. This makes me think it cant be due to sensitivyt - If sensitivity was non-proportionally high for the hot probe, surely all my amounts of protein loaded would give a similar (perhaps saturated) band intensity.

For information - this is a biotin-labeled probe detected by Strept-HRP and ECL so yes its chemiluminescence. However, many people use this method and several kits are available to buy which utilize this method - so it must work for some. Im thinking now my probe must be binding non-specifically to my proteins so that nothing is competed away by the cold probe. The only problem is im not sure how to fix it - perhaps modifying the binding buffer.

Anyway, thanks for your ideas



I don't have experience with EMSAs, but maybe it's a detection sensitivity problem? If your cold probe displaces 99% of your labeled probe, would 1% label still show up? Maybe you're asking too much expecting the band to be completely off -- the cold and labeled probe exist in equilibrium, there will always be some label, right? Especially in light of "oddly, the free probe band intensity often decreases noticeably in my competition lanes"...

How are you detecting your signal? If it's qualitative, like chemiluminescence, maybe you need a more quantitative method?






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