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

Mass Spectrometry Forum Discuss and post questions about LC-MS, Q-STAR, MALDI-TOF, and other Mass Spectrometry Questions in the Mass Spec Forum.


In-gel trypsin digestion

In-gel trypsin digestion - Mass Spectrometry Forum

In-gel trypsin digestion - Discuss and post questions about LC-MS, Q-STAR, MALDI-TOF, and other Mass Spectrometry Questions in the Mass Spec Forum.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-16-2009, 06:23 AM
crd crd is offline
Pipette Filler
Points: 58, Level: 1 Points: 58, Level: 1 Points: 58, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default In-gel trypsin digestion



Does anyone have a good, reliable in-gel trypsin procedue, with good recovery from the gel subsequently.

Thanks
Reply With Quote
  #2  
Old 02-21-2011, 08:18 PM
Pipette Filler
Points: 331, Level: 6 Points: 331, Level: 6 Points: 331, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2011
Location: Morgantown, WV
Posts: 7
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: In-gel trypsin digestion

Hey crd,

The following is a good reliable [Only registered users see links. ]


As an alternate for extraction protocol after overnight digestion I have centrifuged and collected the supernatant in a clean tube. Dehydrated the gel pieces with 50 μL 100% Acetonitrile and incubate at RT for 20 mins. Pooled together both supernatants in the same tube and dried down with Lyophilizer or SpeedVac. Once dry, I typically recommended an acetic acid clean-up (50 μL of 0.1M Glacial Acetic Acid). Dry down in lyophilizer or SpeedVac.

If this is for mass spec. analysis I would recommend clean-up of the salts with [Only registered users see links. ] or [Only registered users see links. ].

Good luck!

Pamela Williams
Laboratory Technician at Protea Biosciences
[Only registered users see links. ]

Last edited by proteaPam; 03-24-2011 at 02:49 PM.
Reply With Quote
  #3  
Old 07-20-2011, 06:23 AM
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: In-gel trypsin digestion

Yup. That is a pretty standard in gel digest procedure. I like SepPak columns from waters for cleanup but it they might be too much for lowly abundant proteins in a gel band.
Reply With Quote
Reply

Tags
digestion , ingel , trypsin


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
Restriction Enzyme Digestion Protocol admin DNA Techniques 1 04-13-2011 06:11 AM
Scaling-up of partial digestion waikhay Protocols and Methods Forum 1 08-14-2007 02:05 AM
Quesion about DNA gel digestion Jayakumar, R Protocols and Methods Forum 0 07-31-2007 02:26 PM
Methods Digest, Vol 19, Issue 7 to WS Organ Trypsin digestion Jess Protocols and Methods Forum 0 12-09-2006 06:20 AM
trypsin digestion nithya Protein Forum 1 12-17-2005 06:07 PM


All times are GMT. The time now is 05:25 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 0.13205 seconds with 16 queries