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

Protein Science Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


SDS-PAGE Protocol

SDS-PAGE Protocol - Protein Science

SDS-PAGE Protocol - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


Reply
 
LinkBack Thread Tools Display Modes
  #11  
Old 01-14-2009, 02:41 PM
Pipette Filler
Points: 63, Level: 1 Points: 63, Level: 1 Points: 63, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: SDS-PAGE Protocol

Thank u very much for delivering such a nice protocol
Reply With Quote
  #12  
Old 02-09-2010, 01:56 AM
Pipette Filler
Points: 4, Level: 1 Points: 4, Level: 1 Points: 4, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2010
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: SDS-PAGE Protocol

@akj - it could be that the amount of p24 in the gel is not detectable with the staining method you are using. Silver staining is more sensitive than coomassie staining, of course there are other staining techniques that you may want to explore also. If it is a particularly low abundance protein i'd be looking to employ antibodies.
Reply With Quote
  #13  
Old 02-11-2010, 05:30 PM
Pipette Filler
Points: 67, Level: 1 Points: 67, Level: 1 Points: 67, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2010
Location: oswestry
Posts: 3
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: SDS-PAGE Protocol

hi,
i'm new to this forum and i'm not experience in western blotting.
how to minimise background for western blot analysis. i use monoclonal antibodies
first i:
- block with casein milky solution( 1gram milk + 25ml of Incubation buffer-IB) for 1hrs
-then wash x3 with PBS to remove the milk
-add primary anti body
-leave on rocker for 1hr
-wash x3 with IB then x3 with PBS
- add secondary antibody usualy anti-mouse HRP
-leave for 1hr on rocker
- wash x2 with IB then x3 with PBS to remove the incubation buffer
- completly remove all left over liquide
- add substrate (west femto)
-leave for 5min
- analyse result on chemiluminocent (biorad)

this is my normal routine but i always get backgroud or low bands
is it anyway to improuve my protocol to avoid backgroung
i will apreciate any advice...
just want a publishable quality images.
thanks again for the other information on western blot protocol .. i'm getting very good blot since i red your protocol.




and i wash x2 with icubation buffer then x3 with PBS
Reply With Quote
  #14  
Old 02-11-2010, 05:36 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Post Re: SDS-PAGE Protocol

Quote:
Originally Posted by anoopbal View Post
Thanks for the protocol.

What is the benifit of staining the gel using commasie blue? I am assuming unless you use aan antibody for a specific protein you cannot differentiate your band from other bands.

Thanks
You can't see the protein bands without staining.
Coomassie blue staining of a gel shows you the bands.
Reply With Quote
  #15  
Old 02-12-2010, 11:27 AM
Pipette Filler
Points: 67, Level: 1 Points: 67, Level: 1 Points: 67, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2010
Location: oswestry
Posts: 3
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: SDS-PAGE Protocol

Quote:
how to test antibodies on western blot?
i have 5 antibodies i would like to test on muscle extract run on 12% gel. what is the best protocol to get a clair result.
thank
Reply With Quote
Reply

Tags
protocol , sdspage


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
Matrigel Invasion Assay Protocol moleculardude Cell Biology and Cell Culture 6 06-11-2012 12:38 PM
Protocol for human CD38 WB Morales, Victor Protein Forum 0 02-02-2008 09:30 PM
Protocol: Inner and Outre membrane Isolation?! Natalia Oliveira Protocols and Methods Forum 1 10-09-2007 10:31 PM
Flow Chamber Assay Protocol moleculardude Cell Biology and Cell Culture 0 11-29-2006 01:02 AM
FS: Old Physics-Related Books Oldbooks78 Physics Forum 0 08-27-2003 02:32 PM


All times are GMT. The time now is 02:19 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13791 seconds with 16 queries