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.


GAPDH Not visible on western blot

GAPDH Not visible on western blot - Western Blot Forum

GAPDH Not visible on western blot - 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 06-30-2008, 02:22 PM
Pipette Filler
Points: 435, Level: 8 Points: 435, Level: 8 Points: 435, Level: 8
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2008
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default GAPDH Not visible on western blot



Hi,

I am trying to detect GAPDH in prostate tissue and its not visible. Tried a variety of Ab concs and also different blockers but still no joy. I'm not sure if there are large amounts of GAPDH protein expressed in prostate tissue, does anyone know if there is and also what other loading controls are good to use? I'm going to try alpha tubulin but any other ideas would be good.
Thanks!
Reply With Quote
  #2  
Old 07-02-2008, 02:48 PM
Volunteer
Points: 515, Level: 10 Points: 515, Level: 10 Points: 515, Level: 10
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2008
Location: NC Illinois
Posts: 40
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: GAPDH Not visible on western blot

Have you been able to detect any other bands on Westerns using the same samples?
What are your antibody concentrations/dilutions and the substrate being used?
Reply With Quote
  #3  
Old 07-03-2008, 03:13 AM
ethidiumbromide's Avatar
Pipette Filler
Points: 459, Level: 9 Points: 459, Level: 9 Points: 459, Level: 9
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2008
Posts: 18
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: GAPDH Not visible on western blot

Assuming that prostate tissue has no detectable levels of GAPDH, maybe try actin?
Reply With Quote
  #4  
Old 10-02-2008, 03:13 PM
Pipette Filler
Points: 275, Level: 5 Points: 275, Level: 5 Points: 275, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2008
Posts: 3
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: GAPDH Not visible on western blot

ethidiumbromide has the right idea. I have found that Beta-actin is very reliable. I use it at about a 1:10,000 concentration.

I have also been having troubles with my GAPDH as of late. After searching the web, I have learned that GAPDH will not reliably band when blocked with anything other than a 5% BSA.

Hope that helped a bit, Good luck!
Reply With Quote
The Following User Says Thank You to anna.oskar For This Useful Post:
mbsum09 (07-17-2009)
  #5  
Old 07-17-2009, 08:26 PM
Pipette Filler
Points: 22, Level: 1 Points: 22, Level: 1 Points: 22, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 1
Thanks: 1
Thanked 1 Time in 1 Post
Default Re: GAPDH Not visible on western blot

GAPDH has likely run off the gel. Try running the gel for less time.
Reply With Quote
The Following User Says Thank You to mbsum09 For This Useful Post:
admin (07-17-2009)
Reply

Tags
blot , gapdh , visible , western


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
Western Blot Background Troubleshooting molecule2005 Western Blot Forum 18 10-07-2010 11:49 AM
Can we use dot blot inplace of Western blot? peterish Western Blot Forum 3 06-16-2010 07:59 PM
Western blot admin Article Discussion 2 04-23-2010 07:31 PM
dot blot and western blot Liang Western Blot Forum 1 08-07-2009 03:26 PM
western blot question Rebecca Pickin Protocols and Methods Forum 2 07-25-2007 08:01 PM


All times are GMT. The time now is 07:52 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 0.13483 seconds with 16 queries