Re: silver stain

From: Ken Mitchelhill (ken_mitchelhill@hotmail.com)
Date: Tue May 29 2001 - 19:31:28 EDT

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<DIV>Hi Jeff,</DIV>
<DIV> </DIV>
<DIV>it seems you are not the only one with problems accessing
Gary's protocol, I have taken the liberty of sending a copy to the ABRF list as
I expect there will be a few others out there interested in seeing this very
easy protocol. I have sent it in HTML format and apologise in advance to those
with plain text email clients.</DIV>
<DIV> </DIV>
<DIV>Regards....Ken</DIV>
<DIV> </DIV>
<DIV>
<BLOCKQUOTE>
 <BLOCKQUOTE>
 <A name=top></A>Silver staining
 of proteins in polyacrylamide gels</BLOCKQUOTE></BLOCKQUOTE>
<BLOCKQUOTE>
 <BLOCKQUOTE>
 (adapted from Blum et
 al. Electrophoresis, 8, 93-99, 1987) </BLOCKQUOTE></BLOCKQUOTE>
<HR width="90%" color=#a3a5d3>

<BLOCKQUOTE>
 <BLOCKQUOTE>
 Everything
 <BLINK>Fresh!</BLINK>
 
 This is an easy staining protocol
 and is compatible with mass spectrometric protein analysis. The stain is
 useful for protein concentrations ranging from <1ug to >1ng. If the
 staining does not work, for whatever reason, then gels can easily be
 <A
 href="http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining.html#Destaining protocol">destained</A> and restained again (see below). </BLOCKQUOTE></BLOCKQUOTE>
<A
href="http://gimr.garvan.unsw.edu.au/public/corthals/guidelines/guidelines_ext.html"><IMG height=25
src="http://www.garvan.unsw.edu.au/public/corthals/guidelines/dotL.gif" width=14
align=textTop border=0></A> <A
href="http://gimr.garvan.unsw.edu.au/public/corthals/guidelines/guidelines_ext.html">guidelines</A> <IMG height=25
src="http://www.garvan.unsw.edu.au/public/corthals/guidelines/dotL.gif" width=14
align=textTop> <A
href="http://www.garvan.unsw.edu.au/public/corthals/">home</A>
<HR width="90%" color=#a3a5d3>

<BLOCKQUOTE>
 <BLOCKQUOTE>
 <H2>Gel staining
 protocol</H2></BLOCKQUOTE></BLOCKQUOTE>
<DIV align=center>
<CENTER>
<TABLE cellSpacing=0 cellPadding=3 border=1>
 <TBODY>
 <TR>
 <TD width=20 height=16> </TD>
 <TD width=80 height=16>solution</TD>
 <TD width=300 height=16> </TD>
 <TD width=125 height=16>V =100 ml</TD>
 <TD width=125 height=16>operation</TD>
 <TD width=125 height=16>time</TD></TR>
 <TR>
 <TD width=20 height=16>1</TD>
 <TD width=80 height=16>sol
 A</TD>
 <TD width=300 height=16>50% methanol 10% acetic acid</TD>
 <TD width=125 height=16> </TD>
 <TD width=125 height=16>fix</TD>
 <TD width=125 height=16>30
 min</TD></TR>
 <TR>
 <TD width=20 height=16>2</TD>
 <TD width=80 height=16>sol
 B</TD>
 <TD width=300 height=16>5%
 methanol</TD>
 <TD width=125 height=16> </TD>
 <TD width=125 height=16>incubate</TD>
 <TD width=125 height=16>15
 min</TD></TR>
 <TR>
 <TD width=20 height=16>3</TD>
 <TD width=80 height=16> </TD>
 <TD width=300 height=16>milli-Q H2O</TD>
 <TD width=125 height=16> </TD>
 <TD width=125 height=16>wash 3 times</TD>
 <TD width=125 height=16>3
 x 5 min</TD></TR>
 <TR>
 <TD width=20 height=16>4</TD>
 <TD width=80 height=16>sol. C</TD>
 <TD width=300 height=16><A name="solution c"></A>sodiumthiosulphate (Na2S2O3.5H2O)</TD>
 <TD width=125 height=16>0.2 g/L fresh!</TD>
 <TD width=125 height=16>Incubate</TD>
 <TD width=125 height=16>120 sec</TD></TR>
 <TR>
 <TD width=20 height=16>5</TD>
 <TD width=80 height=16> </TD>
 <TD width=300 height=16>milli-Q H2O</TD>
 <TD width=125 height=16> </TD>
 <TD width=125 height=16>wash 3 times</TD>
 <TD width=125 height=16>3
 x 30 sec</TD></TR>
 <TR>
 <TD width=20 height=16>6</TD>
 <TD width=80 height=16>sol
 D</TD>
 <TD width=300 height=16>silver nitrate (AgNO3)</TD>
 <TD width=125 height=16>0.2 g/100 ml</TD>
 <TD width=125 height=16>Incubate</TD>
 <TD width=125 height=16>25
 min</TD></TR>
 <TR>
 <TD width=20 height=16>7</TD>
 <TD width=80 height=16> </TD>
 <TD width=300 height=16>milli-Q H2O</TD>
 <TD width=125 height=16> </TD>
 <TD width=125 height=16>wash 3 times</TD>
 <TD width=125 height=16>3
 x 60 sec</TD></TR>
 <TR>
 <TD width=20 height=16>8</TD>
 <TD width=80 height=16>sol
 E</TD>
 <TD width=300 height=16>sodium carbonate (Na2CO3) 37% HCOH Na2S2O3.5H2O (sol.C)</TD>
 <TD width=125 height=16>3
 g/100 ml 50 ul/100 ml 2 ml/100 ml</TD>
 <TD width=125 height=16>develop</TD>
 <TD width=125 height=16>10
 min max</TD></TR>
 <TR>
 <TD width=20 height=16>9</TD>
 <TD width=80 height=16>sol
 F</TD>
 <TD width=300 height=16>Na2-EDTA </TD>
 <TD width=125 height=16>(14g / L)</TD>
 <TD width=125 height=16>stop develop</TD>
 <TD width=125 height=16>10
 min</TD></TR>
 <TR>
 <TD width=20 height=16>10</TD>
 <TD width=80 height=16> </TD>
 <TD width=300 height=16>milli-Q H2O</TD>
 <TD width=125 height=16> </TD>
 <TD width=125 height=16>wash</TD>
 <TD width=125 height=16> </TD></TR></TBODY></TABLE></CENTER></DIV>
 
<A
href="http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining.html#top"><IMG height=12
src="http://www.garvan.unsw.edu.au/public/corthals/guidelines/top.gif" width=12
border=0></A>
<HR width="90%" color=#a3a5d3>

<BLOCKQUOTE>
 <BLOCKQUOTE>
 <A name="Destaining protocol"></A>Destaining protocol
 <UL>
 <LI>Dissolve 0.4g potassium
 ferricyanide (K3Fe(CN)6) in 200ml
 sodiumthiosulphate
 (0.2 g/L, <A
 href="http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining.html#solution c">solution c</A> above). </LI></UL>
 <BLOCKQUOTE>
 Destain: 
 <UL>
 <LI>destain until no bands are
 visible; the gel will have a yellow hue!
 <LI>wash gel 4-5 times for 15 min
 with milli-Q
 H2O until gel is
 transparent and has no background colour.
 <LI>stain gel again starting with
 <A
 href="http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining.html#solution c">solution c</A> from gel staining protocol.
 </LI></UL></BLOCKQUOTE></BLOCKQUOTE></BLOCKQUOTE>
<A
href="http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining.html#top"><IMG height=12
src="http://www.garvan.unsw.edu.au/public/corthals/guidelines/top.gif" width=12
border=0></A>
<HR width="90%" color=#a3a5d3>
</DIV>
<DIV> </DIV>
<DIV>
01011999 updated Garry
Corthals (<A href="mailto:g.corthals@garvan.unsw.edu.au">g.corthals@garvan.unsw.edu.au</A>) 
 </DIV>
<DIV>----- Original Message ----- 
<DIV>From: "Jeffrey A Kowalak, Ph.D." <<A
href="mailto:jkowalak@intra.nimh.nih.gov">jkowalak@intra.nimh.nih.gov</A>></DIV>
<DIV>To: "Ken Mitchelhill" <<A
href="mailto:ken_mitchelhill@hotmail.com">ken_mitchelhill@hotmail.com</A>></DIV>
<DIV>Sent: Tuesday, May 29, 2001 8:08 PM</DIV>
<DIV>Subject: RE: silver stain</DIV></DIV>
<DIV> </DIV>> Hello Ken, > >
Apparently the link you posted is accessible only to internal UNSW
users. > Any chance of copying and pasting the protocol? >
Regards, > Jeff > > Jeffrey A Kowalak, Ph.D. > Mass
Spectrometry Facility Director > Laboratory of Neurotoxicology >
National Institute of Mental Health > Building 10 Room 3D42 >
Bethesda, MD 20892-1262 > > Telephone: 301-496-4242 >
Facsimile: 301-480-0198 > e-mail: <A
href="mailto:jkowalak@intra.nimh.nih.gov">jkowalak@intra.nimh.nih.gov</A> > >
-----Original Message----- > From: Association of Biomolecular Resource
Facilities > [mailto:abrf-request@aecom.yu.edu]On Behalf Of Ken
Mitchelhill > Sent: Thursday, May 24, 2001 7:15 PM > To: Recipients
of ABRF List > Subject: Re: silver stain > > >
Paul, > > Gary Corthals has got a nice protocol at: >
 > <A
href="http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining">http://www.garvan.unsw.edu.au/public/corthals/HTML-protocols/silver_staining></A> > .html > > Ken > ----- Original Message
----- > From: "pds" <<A
href="mailto:pds@fossil.biochem.ualberta.ca">pds@fossil.biochem.ualberta.ca</A>> > To:
"Recipients of ABRF List" <<A href="mailto:abrf@aecom.yu.edu">abrf@aecom.yu.edu</A>> > Sent: Friday, May
25, 2001 4:51 AM > Subject: silver stain > > > >
Hi, > > > >   What is the best prodedure for silver
staining gels for later in gel > digests > > and mass
analysis.Thanks. > > > > Paul Semchuk > > >
> > > </BODY></HTML>

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