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.


Mystery in Western

Mystery in Western - Western Blot Forum

Mystery in Western - 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 08-08-2009, 04:15 AM
Pipette Filler
Points: 1,005, Level: 18 Points: 1,005, Level: 18 Points: 1,005, Level: 18
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 27
Thanks: 0
Thanked 2 Times in 2 Posts
Default Mystery in Western



Hi guys,
Any ideas of what is the problem?

I did 4 western blots, with identical results.
I load just one sample – 20 ug of protein per lane with 1:2 buffer in 8 lanes.
So the expression and quantification should be similar.

However, I keep getting reproducible results for all of the blots – last two lanes have less protein expression and different value for fractions protein/loading control. So, my quantification numbers (fractions) look like 5,6,6,5,6,6,10,11.

At first I thought I have a pipetting issue, but when I got the 4th blot with the same, different expression in the same last 2 lanes I started to think there must be something else. And it can’t be Bradford either because it is one sample!

Any ideas to this mystery?

Thanks!
Reply With Quote
  #2  
Old 08-08-2009, 09:47 PM
Pipette Filler
Points: 1,005, Level: 18 Points: 1,005, Level: 18 Points: 1,005, Level: 18
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 27
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: Mystery in Western

To add - do you think this can be attributed to the leaking in the gel?

We have a bio-rad equipment, and I noticed that gels leak just a bit when I was making them, but I never thought that could result in a problem.
May be leaking is extended and causes uneven gel polymerazation?
Reply With Quote
  #3  
Old 09-08-2009, 09:27 PM
Pipette Filler
Points: 152, Level: 3 Points: 152, Level: 3 Points: 152, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 8
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: Mystery in Western

you guys should try chembio's precast gel system. Its a horizontal SDS PAGE sstem which gaurantees no leaks and simplfies the whole process
Reply With Quote
  #4  
Old 09-10-2009, 12:02 AM
Pipette Filler
Points: 1,005, Level: 18 Points: 1,005, Level: 18 Points: 1,005, Level: 18
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 27
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: Mystery in Western

He-he.

Don't think that will work :-)

But yes, as a matter of fact it was the leaking gels.

So now I just do a 1-2 mm layer of gel with high APS and timed at the bottom of the gels, and only after that pour the resolving gels.

They dont leak and no more funny bands.
Reply With Quote
Reply

Tags
mystery , 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 secreted protein control alphabetty Western Blot Forum 4 02-20-2011 01:33 PM
Western Blot Background Troubleshooting molecule2005 Western Blot Forum 18 10-07-2010 11:49 AM
Western Blot Mystery - Developed bands don't adhere to membrane cyrus Western Blot Forum 4 01-20-2009 09:08 PM
western blot mystery Protocols and Methods Forum 1 12-08-2004 04:49 PM
western blot mystery! Protocols and Methods Forum 0 12-08-2004 02:17 PM


All times are GMT. The time now is 07:47 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 0.12967 seconds with 16 queries