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.


Loading problems - please help

Loading problems - please help - Western Blot Forum

Loading problems - please help - 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 05-12-2011, 10:47 AM
Pipette Filler
Points: 252, Level: 5 Points: 252, Level: 5 Points: 252, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2011
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Loading problems - please help



Hi! I have several years of experience with Western blot and have recently developed issues with variable protein loading.

Briefly, I am using lysates from cultured cells using either a RIPA buffer or a tris-based lysis buffer for phospho-proteins. I am using pre-cast gels and buffers (invitrogen). I have used both a Bradford (Bio-Rad) and Lowry (homemade buffers) for protein determination and run my standards and samples in duplicate so I can average the results. I don't think I am losing the sample during loading as I have not previous had this problem and think my technique is okay.

One big change in my method has been the preparation and reading of the protein concentration. I was preparing my Bradford samples in disposable cuvettes for a spectrophotometer that broke a while back. I adapted the protocol and now prepare the samples in microfuge tubes, place samples into wells on a standard 96 well plate and read the results on a Polar star Omega plate reader.

I can't identify any obvious problems, but my tubulin control blots are ugly and show clear differences in between lanes.

Any ideas?

Thanks!
KiKi
Reply With Quote
Reply

Tags
loading , problems


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
Plasmid-stability? Fragment-deletion? I'm in dire need to identify and solve problems TGS Molecular Cloning Forum 3 10-22-2012 09:17 PM
Loading problems - please help kforema Western Blot Forum 0 05-12-2011 10:53 AM
problems with growing healthy Arabidopsis plants Monika Kuzma Arabidopsis and Plant Biology 1 02-19-2007 03:55 PM
Problems with growing healthy Arabidopsis plants - light intesity? Karl Lundy Arabidopsis and Plant Biology 0 02-07-2007 05:11 PM


All times are GMT. The time now is 06:38 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 0.11449 seconds with 16 queries