Silver stained gels - background problem when using DTT

I am having a small issue with my silver stained gels. (Mini gels, 1.5mm,
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) in
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

"Amanda" <[Only registered users see links. ].uk> wrote in message
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in
High backgrounds in Ag-stain are often attributed to the low purity of water
and other reagents. Also, try presoaking you gels in any DTT-free buffer, or
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 whole
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?
Emir

Amanda wrote:



Which staining protocoll are you using? Have you tried Heukeshoven &
Dernik? In my hands that gives the best results from all the methods I
tried.

Also make sure that all your reagents are scrupoulously clean.

You may consider replacing silver staining with fluorescent staining
(nile red or one of Molecular Probes Sypro stains). They are almost as
sensitive as silver.

I am not sure of the name of the protocol (I'll look it up and get back to
you), but I soak my glassware in HCl to clean them, and scrub them very
carefully, and use only MilliQ-Plus distilled water (22um filter).

I would like to try flourescent protocols, but we don't have a GelDoc system
that can capture fluorescent results very well - I've had a devil of a time
with a FITC-labeled gelatin gel - background is too high, no matter what I
do, and our poor little GelDoc is a simple closed-circuit camera with an
EtBr filter attached.

Can you give me more details on your protocol? Thanks


"Dr Engelbert Buxbaum" <[Only registered users see links. ]> wrote in message
news:btmlqc$rrl$04$[Only registered users see links. ]...
1.5mm,
in

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" <[Only registered users see links. ]> wrote in message
news:vqmKb.15$[Only registered users see links. ]...
1.5mm,
water
or
whole

Buy the way, you could replace BME and DTT with a more powerful reducing
agent TCEP that you can buy e.g. from Pierce. TCEP is 20 times more powerful
than DTT, if I remember that correctly, so you need less of it in the
loading buffer.
Emir

"Cynthia Gillespie" <[Only registered users see links. ].za> wrote in message
news:[Only registered users see links. ].za...
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