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Eluting from magnetic beads in ChIP.


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#1 Trust

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Posted 05 May 2010 - 10:07 AM

Hey there,

First poster. I searched around to see if this particular question had been asked and didn't see anything, so if I'm forum cluttering unknowingly I apologize.

I'm working on a chip protocol for a TF that I've been getting highly variable results from.

I'm using this protocol: http://knol.google.c...-chip-protocol#

My primary is a rabbit anti-mouse, and while we had some limited (highly variable) success with protein g we seemed to have no success getting low backround with protein a, so we're going to take a whack at it using some goat anti-rabbit IgG dynabeads.

I was wondering if I can retain the chelex-100 method for obtaining my DNA if I'm using magnetic beads instead of the agarose? Or what methods people are using to elute from their beads? I was kind of hoping to avoid the sds elution buffers.

Also, if anyone has any general pointers they wish they'd known when they first started doing chip, I'll take them too. I'm in a bit of a time crunch and can't troubleshoot the protocol as thoroughly as I'd like to.

Thanks in advance!

#2 KPDE

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Posted 05 May 2010 - 07:46 PM

Hey there,

First poster. I searched around to see if this particular question had been asked and didn't see anything, so if I'm forum cluttering unknowingly I apologize.

I'm working on a chip protocol for a TF that I've been getting highly variable results from.

I'm using this protocol: http://knol.google.c...-chip-protocol#

My primary is a rabbit anti-mouse, and while we had some limited (highly variable) success with protein g we seemed to have no success getting low backround with protein a, so we're going to take a whack at it using some goat anti-rabbit IgG dynabeads.

I was wondering if I can retain the chelex-100 method for obtaining my DNA if I'm using magnetic beads instead of the agarose? Or what methods people are using to elute from their beads? I was kind of hoping to avoid the sds elution buffers.

Also, if anyone has any general pointers they wish they'd known when they first started doing chip, I'll take them too. I'm in a bit of a time crunch and can't troubleshoot the protocol as thoroughly as I'd like to.

Thanks in advance!


Two people in my building are using the chelex method with magnetic beads successfully.

#3 Mighty Mouse

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Posted 07 May 2010 - 04:42 AM

For what it's worth I've found there to be a bit of a difference in my variability depending on the companies magnetic beads I use. I was using Millipore's protein A beads, but they had supply problems so I started playing with dynabeads from Invitrogen, beads from Cell Signaling and beads from Activ Motif. I elute with 0.1M NaHCO3 + 1% SDS plus proteinase K (incubate 2hrs at 62C with shaking). From my limited experience with one TF (CREB) I got the least amount of variability, by far, with the Millipore protein A beads. So I decided to wait until I finally got those beads in and have been finally moving forward and keeping a large stock of beads around so I don't have to wait around again. But if you're in a time crunch you probably don't want to run around checking beads from various companies...just a thought.

MM
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#4 jamessmith01

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Posted 26 May 2010 - 07:15 AM

@Mighty Mouse:

Interesting info - how did the Active Motif beads fare in your comparison? I'm using the Active Motif ChIP-IT express magnetic kit, but I haven't compared it to any other kits/beads.

#5 Mighty Mouse

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Posted 27 May 2010 - 10:39 AM

@Mighty Mouse:

Interesting info - how did the Active Motif beads fare in your comparison? I'm using the Active Motif ChIP-IT express magnetic kit, but I haven't compared it to any other kits/beads.


James,

They worked pretty well for me just plugging them into my protocol. The data seemed a little bit noisier to me, but it's tough to say because I only ran 2-3 animals for comparison. For whatever reason the beads I had the biggest issue getting to work consistently were those from Invitrogen, not really sure why...

for what it's worth, I started with the Millipore kit and switched to all homemade ingredients and I get much better enrichment; my guess it's primarily due to the differences in elution buffer, but I don't know what Millipore's elution buffer is.

MM
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#6 smed

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Posted 15 June 2010 - 07:40 PM

@Mighty Mouse:

Interesting info - how did the Active Motif beads fare in your comparison? I'm using the Active Motif ChIP-IT express magnetic kit, but I haven't compared it to any other kits/beads.


James,

They worked pretty well for me just plugging them into my protocol. The data seemed a little bit noisier to me, but it's tough to say because I only ran 2-3 animals for comparison. For whatever reason the beads I had the biggest issue getting to work consistently were those from Invitrogen, not really sure why...

for what it's worth, I started with the Millipore kit and switched to all homemade ingredients and I get much better enrichment; my guess it's primarily due to the differences in elution buffer, but I don't know what Millipore's elution buffer is.

MM


hi MM,
would you mind sharing your buffer recipes (there are so many available, and for the beginner like me its difficult to chose which one to go with). i'm currently using Millipore's EZ-Magna chip kit, but still optimising, so 22 appliactions which they provide doesn't give you much room to play around :D
sandra

#7 Mighty Mouse

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Posted 17 June 2010 - 09:14 AM

Sure thing...as best I know these are based off of the original Upstate ChIP protocol. Like I said, I think the only difference between the recipes that I use and those of Millipore's EZ ChIP kit is the elution buffer, and the one I've been using seems to yield more DNA thereby giving me a less noisy signal for my qPCR.

Also, my experiments are done in vivo from tissue removed from animals, so some of the buffers in the beginning of the protocol might be different.

Recipes:

Cell Lysis Buffer
10 mM Tris-HCl (pH 8.1)
10 mM NaCl
1.5 mM MgCl2
0.5 % Igepal-CA630

Nuclear Lysis Buffer
50 mM Tris-HCl (pH 8.1)
5 mM EDTA
1% SDS

Low Salt Wash Buffer
0.1% SDS
1.0% Triton X-100
2 mM EDTA
20 mM Tris-HCl (pH 8.1)
150 mM NaCl

High Salt Wash Buffer
0.1% SDS
1.0% Triton X-100
2 mM EDTA
20 mM Tris-HCl (pH 8.1)
500 mM NaCl

LiCl Wash Buffer
1.0% Igepal-CA630
1.0% deoxycholate
1 mM EDTA
10 mM Tris-HCl (pH 8.1)
250 mM LiCl

TE Buffer
10 mM Tris-HCl (pH 8.1)
1 mM EDTA

Dilution Buffer
16.7 mM Tris-HCl (pH 8.1)
1.1% Triton X-100
0.01 % SDS
167 mM NaCl

Elution Buffer
1% SDS
0.1M NaHCO3¬


For protease/phosphatase inhibitor I use a cocktail from Pierce.

I hope that helps!

MM
We are all artists...painting with experience on the canvas of life

#8 smed

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Posted 20 June 2010 - 05:13 PM

Sure thing...as best I know these are based off of the original Upstate ChIP protocol. Like I said, I think the only difference between the recipes that I use and those of Millipore's EZ ChIP kit is the elution buffer, and the one I've been using seems to yield more DNA thereby giving me a less noisy signal for my qPCR.

Also, my experiments are done in vivo from tissue removed from animals, so some of the buffers in the beginning of the protocol might be different.

Recipes:

Cell Lysis Buffer
10 mM Tris-HCl (pH 8.1)
10 mM NaCl
1.5 mM MgCl2
0.5 % Igepal-CA630

Nuclear Lysis Buffer
50 mM Tris-HCl (pH 8.1)
5 mM EDTA
1% SDS

Low Salt Wash Buffer
0.1% SDS
1.0% Triton X-100
2 mM EDTA
20 mM Tris-HCl (pH 8.1)
150 mM NaCl

High Salt Wash Buffer
0.1% SDS
1.0% Triton X-100
2 mM EDTA
20 mM Tris-HCl (pH 8.1)
500 mM NaCl

LiCl Wash Buffer
1.0% Igepal-CA630
1.0% deoxycholate
1 mM EDTA
10 mM Tris-HCl (pH 8.1)
250 mM LiCl

TE Buffer
10 mM Tris-HCl (pH 8.1)
1 mM EDTA

Dilution Buffer
16.7 mM Tris-HCl (pH 8.1)
1.1% Triton X-100
0.01 % SDS
167 mM NaCl

Elution Buffer
1% SDS
0.1M NaHCO3¬


For protease/phosphatase inhibitor I use a cocktail from Pierce.

I hope that helps!

MM


much apreciated, thanks.
sandra

#9 dennyG

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Posted 25 August 2010 - 11:11 PM

For what it's worth I've found there to be a bit of a difference in my variability depending on the companies magnetic beads I use. I was using Millipore's protein A beads, but they had supply problems so I started playing with dynabeads from Invitrogen, beads from Cell Signaling and beads from Activ Motif. I elute with 0.1M NaHCO3 + 1% SDS plus proteinase K (incubate 2hrs at 62C with shaking). From my limited experience with one TF (CREB) I got the least amount of variability, by far, with the Millipore protein A beads. So I decided to wait until I finally got those beads in and have been finally moving forward and keeping a large stock of beads around so I don't have to wait around again. But if you're in a time crunch you probably don't want to run around checking beads from various companies...just a thought.

MM



hi MM,
I would like to ask about the Millipore magnetic beads you're using for ChIP.
I'm using Millipore EZ-ChIP kit and would like to switch to home-made reagents too.
I think the magnetic bead from the Millipore kit is good, and went to search for the beads in Millipore www.
But it turns out that they only sell "PureProteome™ Magnetic Beads", which, they say, is not recommended for ChIP.
Are you using this "PureProteome™ Magnetic Beads"? Or have I missed somethings on the company webpages?
Thanks for help in advance.

denny

#10 Mighty Mouse

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Posted 26 August 2010 - 04:31 AM

Hi denny,

They do offer the magnetic beads, but only Protein A magnetic beads; as best I can tell they don't offer protein G magnetic beads by themselves for ChIP (only with their magna-ChIP kit). The catalog number for the Protein A beads is 16-661. For what it's worth when I'm dealing with antibodies that bind protein G better than protein A I use the ActivMotif ChIP-it protein G beads. I just slot them into the protocol the same as I do with the protein A beads from Millipore and it seems to work just fine so far.

hope that helps

MM

Edited by Mighty Mouse, 26 August 2010 - 04:31 AM.

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#11 dennyG

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Posted 26 August 2010 - 10:55 PM

Hi denny,

They do offer the magnetic beads, but only Protein A magnetic beads; as best I can tell they don't offer protein G magnetic beads by themselves for ChIP (only with their magna-ChIP kit). The catalog number for the Protein A beads is 16-661. For what it's worth when I'm dealing with antibodies that bind protein G better than protein A I use the ActivMotif ChIP-it protein G beads. I just slot them into the protocol the same as I do with the protein A beads from Millipore and it seems to work just fine so far.

hope that helps

MM


MM,
This's really a great piece of information. I'll try it out. Thanks so much!!
denny

#12 Steph Kyle

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Posted 02 April 2013 - 03:32 PM

Hi MM,

I stumbled across this thread while hunting for some recipes to make homemade reagents to incorporate into the ChIP kit I am using. I have a quick question:

Do you do SDS elution off your beads AND proteinase K incubation in the same step (62C for 2 hours while shaking)? Any RNase A treatment along with it?


Thank you in advance :) I've read your responses on many threads on this site and they've been very helpful!

Edited by Steph Kyle, 02 April 2013 - 03:34 PM.





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