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

Proteomics Forum Post Questions and Discuss Proteomics, Proteomic Bioinformatics, Proteomic Techniques such as 2-D, Mass Spec etc.


"crashing" with iTRAQ dissolution buffer

"crashing" with iTRAQ dissolution buffer - Proteomics Forum

"crashing" with iTRAQ dissolution buffer - Post Questions and Discuss Proteomics, Proteomic Bioinformatics, Proteomic Techniques such as 2-D, Mass Spec etc.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-17-2011, 07:44 PM
Pipette Filler
Points: 30, Level: 1 Points: 30, Level: 1 Points: 30, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default "crashing" with iTRAQ dissolution buffer



I have peptides product extracted from SDS-PAGE gel after trypsin digestion (tiny amount). Then I try to dissolve them in the iTRAQ buffer (0.5 M TEAB, 30 uL). Whenever I bring the pH to 8.5, the solution turns to "milky". I also tried 0.5 M borate buffer and same thing happens.

I wonder what could've been wrong? In-gel digestion was done with 50 mM TEAB, and peptides were extracted with 60% ACN/5% FA, and dried out ...

Please help. Thanks!
Reply With Quote
  #2  
Old 08-30-2011, 03:04 PM
Pipette Filler
Points: 156, Level: 3 Points: 156, Level: 3 Points: 156, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Location: Palo Alto, CA
Posts: 8
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: "crashing" with iTRAQ dissolution buffer

Actually, I have often noticed precipitate in iTRAQ labeling reactions. I have never tried to resolubilize the the precipitate and analyze it so I am not sure exactly what it is. But I have tested ratios and analyzed the sample and it has never seemed to cause a problem. For instance, if only one of the four samples had a precipitate the ratios still came out as expected. And it didn't seem to effect number of IDs as I was getting similar numbers to those from unlabeled sample. My samples were always whole cell lysates FYI.

While its disconcerting to see, it might not cause you problems in the end.

Also, I would be curious to hear if anyone knows what this is and why it happens.

Cheers,

Doug
Reply With Quote
Reply

Tags
buffer , crashing , dissolution , itraq


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
Transfer problems-smeared blot annie0202 Western Blot Forum 1 02-23-2011 04:10 PM
protein purification magnificientleo Proteomics Forum 11 12-29-2010 09:01 AM
SDS-PAGE Protocol moleculardude Protein Science 14 02-12-2010 11:27 AM
Production of Monoclonal Antibodies - Technique in Mouse Cellbiogal Antibody Forum 2 01-08-2009 10:52 PM
Methods Digest, Vol 18, Issue 19 1. Qiagen buffer PN Jess Protocols and Methods Forum 0 11-22-2006 10:45 PM


All times are GMT. The time now is 10:08 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 0.12584 seconds with 16 queries