Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > General Science Questions and Layperson Board
Register Search Today's Posts Mark Forums Read

General Science Questions and Layperson Board General Science Questions and Layperson Board


Loading controls for neuritogenesis

Loading controls for neuritogenesis - General Science Questions and Layperson Board

Loading controls for neuritogenesis - General Science Questions and Layperson Board


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-29-2012, 08:16 AM
Pipette Filler
Points: 71, Level: 1 Points: 71, Level: 1 Points: 71, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2012
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Loading controls for neuritogenesis



Hi,

I am looking at neurite outgrowth for PC12 and SHSY5Y cells.
I have 2 questions:

1. I am interested in the phosphorylation status of certain genes such as Erk and Creb. Can someone clarify if using the non-phosphorylated proteins as loading controls is sufficient, even at later time points after treatment (24h-72h)?

2. If I want to measure expression of neuronal markers such as beta3 tubulin and GAP43, what loading controls are good? Are there any publications supporting this?

Thanks!
Reply With Quote
  #2  
Old 07-03-2012, 08:35 AM
Pipette Filler
Points: 1,688, Level: 24 Points: 1,688, Level: 24 Points: 1,688, Level: 24
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2010
Posts: 21
Thanks: 0
Thanked 8 Times in 7 Posts
Default Re: Loading controls for neuritogenesis

Hey AA,
as it comes with every phosporylation study you require at least two antibodies for your Western Blot.
First one is an antibody against your phosphorylated protein of interest and the second one against the total protein. So in that case u need anti-phospho Erk and anti-phospho CREB and antibodies against the total Erk and CREB.
Loading simply "unphosphorylated" protein sample and screening with a phospho Ab does not control properly the phorphorylation status of your gene. You need to have the total Ab also.
Reply With Quote
  #3  
Old 07-04-2012, 06:24 AM
Pipette Filler
Points: 71, Level: 1 Points: 71, Level: 1 Points: 71, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2012
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Loading controls for neuritogenesis

Hi Drekketh,

Thank you for your reply
Let me rephrase this to make sure I got it right.
To look at phosphorylation status of Erk, I need:
1. phospho-Erk antibody
2. Total Erk antibody
Do I also need a normal housekeeping gene like GAPDH or actin (especially for time points past 24h)?

Is there a good housekeeping gene I could use for measuring expression of other neurite specific factors such as GAP43 and beta tubulin?

THANKS!!
AA
Reply With Quote
Reply

Tags
controls , loading , neuritogenesis


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 Controls for Membrane Fraction CPF Western Blot Forum 1 02-15-2012 04:28 AM
Loading problems - please help kforema Western Blot Forum 1 12-31-2011 11:39 PM
Loading control that does not register red blood cell proteins synthesize Western Blot Forum 1 10-04-2011 06:03 PM
test if there is a difference between cases and controls SSN BioStatistics Forum 0 12-21-2009 02:38 AM
Loading Controls kiki06 Signalling Biology 4 06-25-2008 07:12 AM


All times are GMT. The time now is 07:44 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 0.12424 seconds with 16 queries