Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > ELISA Assay Forum
Register Search Today's Posts Mark Forums Read

ELISA Assay Forum ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


ELISA Signal from Null-mutant Problem

ELISA Signal from Null-mutant Problem - ELISA Assay Forum

ELISA Signal from Null-mutant Problem - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 05-25-2010, 03:36 PM
Pipette Filler
Points: 754, Level: 15 Points: 754, Level: 15 Points: 754, Level: 15
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2009
Posts: 4
Thanks: 0
Thanked 1 Time in 1 Post
Default ELISA Signal from Null-mutant Problem



Hi all,

I've been developing an sandwich ELISA for an Arabidopsis leaf protein. Below are the details of my procedure so y'all can adequately judge my method. I've just tested my method with the following kinds of samples:

1. An induced control, which shows crisp, clear bands on Western blot consistently
2. Null-mutant extract with 1 ng/uL of target protein added as a control
3. Null-mutant extract as a negative control (and to check for non-target signal)

After the ELISA I get great standard curves. But the null-mutant negative controls show equal or greater signal than the positive controls. Furthermore, I calculate the target concentration that should be 1 ng/uL as around 1.9 ng/uL. I'm really baffled by this and getting fearful my efforts have been for nothing.

Any advice at all would be hugely helpful. Thanks so much in advance!


Here are a few extra details:
----------------------------------------------------------------------
A. I just realized that I personally have never actually run the null-mutant on Western Blot but obviously someone has. However, I've blotted the whole length of the gel plenty of times with both my Primary and Secondary antibody and only very, very occasionally seen extra bands, which have always been only slightly smaller than the target and were interpreted as degraded target.

B. There was a miscalculation in my TBST recipe so that it was 0.01% Tween not the intended 0.2%... could that be a problem?


Below are the details of the procedure:
-----------------------------------------------------------------------
Protein Preparation:
1. Extract leaf proteins with SDS Buffer
2. Precipitate proteins with acetone and re-suspend in PBS (or Triton X-100 Buffer). Re-suspended samples show same level of signal on Western Blot using the same antibodies as in ELISA... single, crisp bands with no other signals at all.

Sandwich ELISA:
1. Coat overnight with 100 ul/well 1:4000 Rabbit polyclonal (recognizes N-terminus of target) primary antibody
2. Wash 3X with 200 ul/well TBST
3. Block overnight with 200 ul/well 5% Non-fat Dry Milk
4. Rotate for 3 hours with 90 ul (normally 100 uL) of sample (note that the protein standard was also added as 90 uL/well)
5. Wash 3X with 200 ul/well TBST
6. Rotate for 2 hours with 100 ul/well 1:1000 Hen polyclonal (recognizes C-terminus of target) secondary antibody labelled with Biotin
7. Wash 3X with 200 ul/well TBST
8. Rotate for 1 hour with 100 ul/well 1:1000 Streptavidin-AP
9. Wash 3X with 200 ul/well TBST
10. Develop with 100 uL ready-to-use pNPP reagent
11. Read at 405 nm!
Reply With Quote
The Following User Says Thank You to WolfeMD For This Useful Post:
admin (05-25-2010)
Reply

Tags
elisa , nullmutant , problem , signal


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
SDS-PAGE problem Steve Nothwehr Protocols and Methods Forum 2 10-16-2009 08:59 AM
Problem with signal detection sharmakaushikp Western Blot Forum 2 08-28-2008 02:33 PM
An unexplanable Tail-PCR problem with Salk mutant lines Guan Yuefeng Arabidopsis and Plant Biology 0 04-20-2006 04:22 AM
Western blot problem -disappearing proteins! Jayakumar, R Protocols and Methods Forum 0 12-13-2004 02:21 PM
FW: SDS-PAGE problem Deanne Bell Protocols and Methods Forum 0 03-05-2004 05:27 PM


All times are GMT. The time now is 02:57 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 0.12488 seconds with 16 queries