Silver stained gels - background problem when using DTT
gillespieza at absamail.co.za
Wed Jan 28 15:35:36 EST 2004
Sorry, I wasn't very clear - the background is uniform across the sample
lanes. There is no background in lanes that have been left empty. Similarly,
there is no background if I prepare sample buffer with BME rather than DTT
(just this awful line of keratins at ~68kDa), so the DTT must be the cause
of the background (this is the only thing that has changed).
I use Milli-Q-Plus water (double distilled, 22 micron filter), which is the
purist water we have in our dept. And as far as the BME is concerned - beats
me! I think its just a poor quality product. But apparently keratins
dissolve in BME incredibly easily, and I've been warned that our supplies
often arrive from the supplier contaminated. I thought I would try DTT
rather than ordering molecular grade BME, but it looks like I should go
ahead and order it anyway though...
"EK" <nobody at elnino.com> wrote in message
news:vqmKb.15$Y4.11258 at news.uchicago.edu...
> "Amanda" <amanda_dreamweaver at yahoo.co.uk> wrote in message
> news:_M-dnexj4fMj5GWiRVn-hA at is.co.za...
> > I am having a small issue with my silver stained gels. (Mini gels,
> > run at 18mA, unlimiting voltage).
> > I use a reducing treatment buffer containing DTT as our supplies of
> > b-mercaptoethanol are badly contaminated with keratins. However, I get a
> > horrible uniform background yellow/brown colour (during the development)
> > the lanes I loaded with this buffer (I've tried just buffer alone).
> > Fortunately, it doesn't seem to come up in the photographs, but it is
> > disturbing, nonetheless.
> > Any suggestions?
> > Amanda
> High backgrounds in Ag-stain are often attributed to the low purity of
> and other reagents. Also, try presoaking you gels in any DTT-free buffer,
> just water, before silver staining. If the DTT is in the sample only, you
> should see a background along the tracks, not uniformly throughout the
> gel. Soaking in a couple of changes of a DTT-free buffer should wash away
> most of DTT (if indeed it is a culprit, which I am not sure).
> Just curious, how come your BME is contaminated with keratins?
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