Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Western Blot Forum
Register Search Today's Posts Mark Forums Read

Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


laemmli buffer dilution

laemmli buffer dilution - Western Blot Forum

laemmli buffer dilution - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-24-2009, 01:36 PM
Pipette Filler
Points: 54, Level: 1 Points: 54, Level: 1 Points: 54, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default laemmli buffer dilution



Hi,

I would be grateful if anyone can help clarify laemmli buffer dilution for me.. I needed less dilution of my protein samples with Laemmli buffer (i usually use 2x Laemmli buffer), so I decided to use 4x laemmli buffer (recipe from molecularstation.com webpage-i cannot post the URL add-new user) the ratio i used was 3:1 (Protein sample:buffer) . However, I noted that the sample seem less blue than the samples receiving 2x Laemmli buffer(is this expected?)

and the recipe mentioned that the concentration of bromophenol to be used is 0.01%, but the final concentration is to be 0.02%. How is this possible?

raya
Reply With Quote
  #2  
Old 09-18-2009, 11:19 PM
Pipette Filler
Points: 657, Level: 13 Points: 657, Level: 13 Points: 657, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2009
Posts: 27
Thanks: 0
Thanked 7 Times in 6 Posts
Default Re: laemmli buffer dilution

I don't think exact and precise concentration of bromophenol blue is a concern I saw a couple recipes simply mentioning to add a tiny pinch to whatever volume is being prepared. I think the only purpose of it is to be able to see the migration front to avoid your proteins being pulled out of the gel during electrophoresis.

The problem I tend to have with 4X Laemmli, is the glycerol precipitating. so I have to shake and vortex it a lot before adding it to my samples.
Reply With Quote
The Following User Says Thank You to Tfal For This Useful Post:
admin (09-19-2009)
  #3  
Old 09-18-2009, 11:20 PM
Pipette Filler
Points: 657, Level: 13 Points: 657, Level: 13 Points: 657, Level: 13
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2009
Posts: 27
Thanks: 0
Thanked 7 Times in 6 Posts
Default Re: laemmli buffer dilution

I don't think exact and precise concentration of bromophenol blue is a concern I saw a couple recipes simply mentioning to add a tiny pinch to whatever volume is being prepared. I think the only purpose of it is to be able to see the migration front to avoid your proteins being pulled out of the gel during electrophoresis.

The problem I tend to have with 4X Laemmli, is the glycerol precipitating. so I have to shake and vortex it a lot before adding it to my samples.
Reply With Quote
  #4  
Old 09-23-2009, 04:39 PM
Pipette Filler
Points: 56, Level: 1 Points: 56, Level: 1 Points: 56, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: laemmli buffer dilution

yes, there is no problem adding a little more bromophenol blue to darken the colour. you can also go up to 5X laemmli buffer [5X:Components: 0.5M Tris-HCl, pH 6.8 6.25ml, SDS 0.5g, Glycerol, 5ml, Bromophenol blue 5mg, Water Up to 10ml] Add 750ul of Laemmli buffer and 250ul of beta mercaptoethanol.
Reply With Quote
Reply

Tags
buffer , dilution , laemmli


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
protein purification magnificientleo Proteomics Forum 11 12-29-2010 09:01 AM
SDS-PAGE Protocol moleculardude Protein Science 14 02-12-2010 11:27 AM
Laemmli buffer admin Article Discussion 1 02-09-2010 01:48 AM
Tris in Laemmli buffer Tfal Western Blot Forum 2 07-08-2009 01:23 PM
Production of Monoclonal Antibodies - Technique in Mouse Cellbiogal Antibody Forum 2 01-08-2009 10:52 PM


All times are GMT. The time now is 04:06 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13436 seconds with 16 queries