Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Troubleshooting with silver staining


  • Please log in to reply
3 replies to this topic

#1 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 28 November 2013 - 03:17 AM

Hi,

 

I follow the quick silver staining protocol-

 

http://www.pangloss....ilverstain.html

 

I am facing lot of problems in this-

  1. It takes lot of time in development, I start to see some bands only after 15 min.
  2. Background is too high.
  3. I add 10% acetic acid to stop the development, as soon as I add this, the faint bands just disappear.

If anyone is using this protocol, please help me in troubleshooting.

 

P.S. I am always using fresh reagents, including DTT

 

 

Thanks



#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,735 posts
125
Excellent

Posted 28 November 2013 - 01:19 PM

our silver stain procedure is a little different than yours (we use a method modified from that of merrill, et al).

 

however, if you add a crystal (small to medium) of sodium thiosulfate to your developer then your background should remain translucent. also, we wash 2x with 1/3 of the developer before developing with the final 1/3. we stop development with 1% acetic acid (actually, we usually use our coomassie destaining solution (30% methanol, 7% acetic acid) so that the gel shrinks back to normal) after removing the developer and washing with water a couple of times (to remove thiosulfate, it doesn't like acid).

 

careful with the water washes after silver incorporation. too much washing will remove too much silver (it is not bound tightly to the protein).

 

i almost forgot, make sure sds removal is as complete as possible, it binds silver like crazy and creates very strong background.


Edited by mdfenko, 28 November 2013 - 01:24 PM.

talent does what it can
genius does what it must
i do what i get paid to do

#3 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 03 December 2013 - 12:39 AM

Thanks mdfenko

 

Like you said that your method is different from our method, earlier we were using vorum's method, where we used to use sodium thiosulfate for sensitization. But in this method, we are not using sodium thiosulfate, instead we are using DTT. I was wondering that principle of both the methods could be different in terms of componentsunsure.png ... therefore I don't know if sodium thiosulfate can be used with this method? 

 

And what do you mean by SDS removal? It should be removed by washes?...DO I need to increase methanol washes?



#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,735 posts
125
Excellent

Posted 03 December 2013 - 03:30 AM

the method we perform uses dichromate to sensitize. we add the thiosulfate to the developer as a clearing agent.

 

we use 10% ethanol/5% acetic acid to wash out sds.


talent does what it can
genius does what it must
i do what i get paid to do




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.