Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Identifying transcription factor binding sites


  • Please log in to reply
6 replies to this topic

#1 yipinglim

yipinglim

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 16 February 2009 - 08:02 PM

Hi,

I'm fairly new to the the ChIP protocols, and am interested in finding out where my protein of interest (which is postulated to be a transcription factor) binds to. From my understanding, if I were to use the EZ ChIP from Upstate, I would need an antibody which binds to my protein of interest, which I do have. However, the last step of the protocol involves using standard PCR or real-time PCR to amplify the region which the protein has bound to.

However, if I do now know where my protein is binding to, how do I design primers to amplify that region?

Basically I would like to find out what are some of the methods that people use to identify unknown binding sites for transcription factors?

Thanks in advance!

#2 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 189 posts
1
Neutral

Posted 17 February 2009 - 01:59 AM

Hi,

I'm fairly new to the the ChIP protocols, and am interested in finding out where my protein of interest (which is postulated to be a transcription factor) binds to. From my understanding, if I were to use the EZ ChIP from Upstate, I would need an antibody which binds to my protein of interest, which I do have. However, the last step of the protocol involves using standard PCR or real-time PCR to amplify the region which the protein has bound to.

However, if I do now know where my protein is binding to, how do I design primers to amplify that region?

Basically I would like to find out what are some of the methods that people use to identify unknown binding sites for transcription factors?

Thanks in advance!


Hey there :P

A while ago I had to look for Pax5 binding sites and I used online software at http://www.gene-regu....com/index.html. Not sure if things have changed since then. Do you know the sequence that your protein binds to in other genes?
Clare

#3 yipinglim

yipinglim

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 18 February 2009 - 12:59 AM

Hi,

I'm fairly new to the the ChIP protocols, and am interested in finding out where my protein of interest (which is postulated to be a transcription factor) binds to. From my understanding, if I were to use the EZ ChIP from Upstate, I would need an antibody which binds to my protein of interest, which I do have. However, the last step of the protocol involves using standard PCR or real-time PCR to amplify the region which the protein has bound to.

However, if I do now know where my protein is binding to, how do I design primers to amplify that region?

Basically I would like to find out what are some of the methods that people use to identify unknown binding sites for transcription factors?

Thanks in advance!


Hey there :lol:

A while ago I had to look for Pax5 binding sites and I used online software at http://www.gene-regu....com/index.html. Not sure if things have changed since then. Do you know the sequence that your protein binds to in other genes?
Clare


Hi Clare,

Thanks for the response.

No, we do not know the sequence that the protein binds to. It's hypothesized to be a transcription factor based on the motifs in the protein, but we have no clue where it binds to. In situations like this, what do people usually do to find out which genes it is regulating at the transcription level?

#4 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 189 posts
1
Neutral

Posted 18 February 2009 - 01:51 AM

Hi again :)

Well, have much money have you got? You could do Chip-seq :lol:

Do you have any ideas at all as to what it binds to? You could design a series of qPCR primers that span a larger region? Apart from that I'm not sure what you could do. All the TFs I work with are pretty well characterised.

Have an awesome day!
Clare

Hi Clare,

Thanks for the response.

No, we do not know the sequence that the protein binds to. It's hypothesized to be a transcription factor based on the motifs in the protein, but we have no clue where it binds to. In situations like this, what do people usually do to find out which genes it is regulating at the transcription level?
[/quote]

#5 jhb80

jhb80

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 18 February 2009 - 02:00 AM

... a comprehensive and probably the easiest (but costly) approach would be a ChIP followed by deep-sequencing (Solexa or 454) of the pulled-down material and map it to the genome to identify associated ORFs.

#6 jhb80

jhb80

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 18 February 2009 - 02:01 AM

wops.... 9 minutes too late :)

#7 yipinglim

yipinglim

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 18 February 2009 - 02:57 AM

Thanks guys. I'll take a look into Chip-seq. That might be exactly what I need.

Yes, the problem is that this protein has not been studied very much at all, so there's really no information on possible binding sites.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.