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 Control Calculations

Loading Control Calculations - Western Blot Forum

Loading Control Calculations - 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 09-22-2009, 10:20 PM
KKM KKM is offline
Pipette Filler
Points: 6, Level: 1 Points: 6, Level: 1 Points: 6, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Loading Control Calculations



I am using Actin as a loading control for my WB. I am running rat brain. I do see slight changes in Actin that I'm confident are not related to our experimental conditions. I expect that they are due errors in protein extraction, concentration analysis and/or loading that are much more prevalent in tissue than in cells, thus making normalizing against loading controls necessary. I want to know how to account for the changes in Actin in my calculations. I have a positive control in all my membranes. Do I make all the wells equal to the positive control for Actin levels and then subtract that factor from my protein of interest density levels, essentially treating it as background? Eg. if my control well Actin density is 20.9, and an experimental well Actin density is 40.8, do i just subtract 19.9 from my experimental well protein of interest density?
Thank you!
Reply With Quote
  #2  
Old 09-23-2009, 01:09 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: Loading Control Calculations

Hi KKM,
Q: are you normalising the protein concentration prior to loading, using a BCA protein assay for eg? Then you will load exactly the same concentration is each well, and therefore the same band intensity should be seen.
I presume that you are restriping the membrane for actin expression, and I usually minus the background signal and divide by actin to give a ratio of test protein expression.
I have also seen that stimulations with some cytokines, such as TNF can alter actin expression and an alternative endogenous control was used.
Reply With Quote
Reply

Tags
actin , calculations , control , loading , loading control , western blot


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
Loading Control montMolec Western Blot Forum 5 11-20-2010 02:14 AM
GADPH Loading Control Western Blot Problem jrscientist Western Blot Forum 3 08-04-2009 06:00 PM
EMSA with tumor samples - Loading control amazonic9 Electrophoretic Mobility Shift Assay Forum 2 09-05-2008 08:10 AM
Loading control? Labnovice Western Blot Forum 1 04-07-2008 03:22 PM
[western blotting] polyclonal vs monoclonal loading control Lechu Protein Forum 1 11-29-2006 09:44 PM


All times are GMT. The time now is 11:16 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 0.11795 seconds with 16 queries