SEQanswers

Go Back   SEQanswers > Applications Forums > Epigenetics



Similar Threads
Thread Thread Starter Forum Replies Last Post
ChIP-Seq with HA-tagged protein Wonko Epigenetics 4 06-24-2014 08:45 AM
BUffers for ChargeSwitch Magnetic Beads ronibras Sample Prep / Library Generation 0 01-13-2012 03:50 AM
ChIP-Seq: ChIP-Seq Data Analysis: Identification of Protein-DNA Binding Sites with SI Newsbot! Literature Watch 0 12-02-2011 04:51 AM
magnetic beads for gel extraction Erika Feltrin Sample Prep / Library Generation 0 03-30-2011 07:39 AM
ChIP-Seq: Systematic characterization of protein-DNA interactions. Newsbot! Literature Watch 0 01-06-2011 03:40 AM

Reply
 
Thread Tools
Old 12-23-2010, 05:31 PM   #1
jdub
Junior Member
 
Location: Central USA

Join Date: Nov 2010
Posts: 8
Default Protein G magnetic beads and ChIP-seq

I am trying to make ChIP work, and my no-antibody control group (chromatin-protein + beads) enriched as much as my positive control (anti-RNA Pol II). Post-IP I washed each ChIP reaction in three different wash buffers a total of six washes. Is this a finesse step? I pipetted beads up and down to mix but not too terribly vigorously.

I was also told that blocking of the beads would not be necessary but maybe it is. Has anyone else found that helpful?
jdub is offline   Reply With Quote
Old 12-24-2010, 12:49 PM   #2
pbluescript
Senior Member
 
Location: Boston

Join Date: Nov 2009
Posts: 224
Default

You can't do a no antibody control with Protein G beads. They will bind a lot of non-specific stuff unfortunately. You'd be better off using an antibody that is specific for something not found in your samples.
pbluescript is offline   Reply With Quote
Old 12-26-2010, 10:55 AM   #3
jdub
Junior Member
 
Location: Central USA

Join Date: Nov 2010
Posts: 8
Default

I use IgG as a negative control as well as a positive control antibody and enrich for a region where it should not amplify by PCR. I see lots of background with both and the no antibody control group tells me that could be a big reason why. So how can I avoid or reduce this, because it appears it will happen in every group regardless of antibody used.
jdub is offline   Reply With Quote
Old 12-26-2010, 11:06 AM   #4
pbluescript
Senior Member
 
Location: Boston

Join Date: Nov 2009
Posts: 224
Default

You can do the standard stuff to try to reduce background like increasing salt concentration or changing the pH of the buffers or increasing the number and volume of the washes. You can also try less beads or switch to protein A beads. I actually had such a bad problem with background with protein G that I switched to the epoxy beads that Invitrogen sells. That fixed my problem.
pbluescript is offline   Reply With Quote
Old 01-17-2011, 07:35 AM   #5
Inti
Member
 
Location: Mexico city

Join Date: Jul 2008
Posts: 12
Default

I usually block the magnetic A or G beads only with BSA for 2 hours and bind the antibody first rather than put the AB in the sample and then the magnetic beads and the ChIPs looks very clean
Inti is offline   Reply With Quote
Old 02-11-2011, 04:58 AM   #6
NGene
Junior Member
 
Location: Germany

Join Date: Feb 2011
Posts: 8
Default

If you do not want to switch to A protein beads, you do not want to use IgG-negative control and you do not want to block the beads, you might want to play with salt concentration and volume/repetitions of the washes.

I had such a bad issue too but I manage to get rid of the background by playing a bit with the above-mentioned parameters. Now I can have 10-fold differences between sample and No-Ab.

I hope it helps.
NGene is offline   Reply With Quote
Reply

Thread Tools

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




All times are GMT -8. The time now is 06:13 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO