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Fast ChIP Method and Chelex - (Feb/19/2008 )

I'm using the Fast ChIP method (or I was, but it wasn't working too well - high background), but when adding Chelex it is hard to add the same amount each time!

Does anyone have tips or tricks for adding equal amounts of this Chelex slurry? It's so variable it drives me nuts. Now I am trying magnetic chip, with a similar dna purification at the end. So again I am using Chelex, but this time in smaller amounts. So the variability drives me nuts.

-MKR-

QUOTE (MKR @ Feb 19 2008, 03:26 PM)
I'm using the Fast ChIP method (or I was, but it wasn't working too well - high background), but when adding Chelex it is hard to add the same amount each time!

Does anyone have tips or tricks for adding equal amounts of this Chelex slurry? It's so variable it drives me nuts. Now I am trying magnetic chip, with a similar dna purification at the end. So again I am using Chelex, but this time in smaller amounts. So the variability drives me nuts.


Hi,

Sorry you're having trouble. When I add the chelex, I vortex each time before adding. Additionally I use a tip with the end cut off since the bead size of the chelex is so large.

As for the background problem, when we developed the assay we were using antibodies to abundant proteins so background wasn't a problem. Now that we've begun looking for proteins that are harder to pull down or at lower abundance on the chromatin we've had to deal with the background problem. A few solutions are:

1) Titrate the amount of chromatin used. If you are using too much, decreasing the amount of chromatin used can improve your signal to background ratio (this has been the most effective technique for us).

2) Block your beads prior to and during the IP with 5% BSA and 100ug/ml sheared salmon sperm DNA (Just make it up in IP buffer with inhibitors).

3) Use more stringent washes (increase salt concentration in later washes).

4) Try decreasing the amount of beads you use (although if you are using the magnetic beads this might not help very much).

Finally, for some proteins that are at low abundance on the chromatin or are difficult to pull down (thus difficult to see above background) we've had the most success with our 96 well plate based ChIP assay, Matrix ChIP (Nucleic Acids Research 2008 36: e17).

In any case, good luck and let me know if you have any other questions.

Joel

-KPDE-

Thankyou, those tips will be very helpful.

What do you mean by block the beads during the IP? I have blocked beads and precleared samples, but do you mean add salmon sperm DNA and BSA with the actual samples?

Oh, as to not get off topic smile.gif is there dry chelex out there? Maybe I could just add dry chelex and that would be more repeatable (I'm mostly worried about the variable amounts of liquid I'm adding which dilute the DNA differently)
.

QUOTE (KPDE @ Feb 20 2008, 11:40 AM)
QUOTE (MKR @ Feb 19 2008, 03:26 PM)
I'm using the Fast ChIP method (or I was, but it wasn't working too well - high background), but when adding Chelex it is hard to add the same amount each time!

Does anyone have tips or tricks for adding equal amounts of this Chelex slurry? It's so variable it drives me nuts. Now I am trying magnetic chip, with a similar dna purification at the end. So again I am using Chelex, but this time in smaller amounts. So the variability drives me nuts.


Hi,

Sorry you're having trouble. When I add the chelex, I vortex each time before adding. Additionally I use a tip with the end cut off since the bead size of the chelex is so large.

As for the background problem, when we developed the assay we were using antibodies to abundant proteins so background wasn't a problem. Now that we've begun looking for proteins that are harder to pull down or at lower abundance on the chromatin we've had to deal with the background problem. A few solutions are:

1) Titrate the amount of chromatin used. If you are using too much, decreasing the amount of chromatin used can improve your signal to background ratio (this has been the most effective technique for us).

2) Block your beads prior to and during the IP with 5% BSA and 100ug/ml sheared salmon sperm DNA (Just make it up in IP buffer with inhibitors).

3) Use more stringent washes (increase salt concentration in later washes).

4) Try decreasing the amount of beads you use (although if you are using the magnetic beads this might not help very much).

Finally, for some proteins that are at low abundance on the chromatin or are difficult to pull down (thus difficult to see above background) we've had the most success with our 96 well plate based ChIP assay, Matrix ChIP (Nucleic Acids Research 2008 36: e17).

In any case, good luck and let me know if you have any other questions.

Joel

-MKR-

I think KPDE means that the slurry contains BSA and Salmon Sperm DNA, and you use this slurry for preclearing and for IP as well

-zek-

QUOTE (MKR @ Feb 20 2008, 09:35 PM)
Thankyou, those tips will be very helpful.

What do you mean by block the beads during the IP? I have blocked beads and precleared samples, but do you mean add salmon sperm DNA and BSA with the actual samples?

Oh, as to not get off topic smile.gif is there dry chelex out there? Maybe I could just add dry chelex and that would be more repeatable (I'm mostly worried about the variable amounts of liquid I'm adding which dilute the DNA differently)
.
QUOTE (KPDE @ Feb 20 2008, 11:40 AM)
QUOTE (MKR @ Feb 19 2008, 03:26 PM)
I'm using the Fast ChIP method (or I was, but it wasn't working too well - high background), but when adding Chelex it is hard to add the same amount each time!

Does anyone have tips or tricks for adding equal amounts of this Chelex slurry? It's so variable it drives me nuts. Now I am trying magnetic chip, with a similar dna purification at the end. So again I am using Chelex, but this time in smaller amounts. So the variability drives me nuts.


Hi,

Sorry you're having trouble. When I add the chelex, I vortex each time before adding. Additionally I use a tip with the end cut off since the bead size of the chelex is so large.

As for the background problem, when we developed the assay we were using antibodies to abundant proteins so background wasn't a problem. Now that we've begun looking for proteins that are harder to pull down or at lower abundance on the chromatin we've had to deal with the background problem. A few solutions are:

1) Titrate the amount of chromatin used. If you are using too much, decreasing the amount of chromatin used can improve your signal to background ratio (this has been the most effective technique for us).

2) Block your beads prior to and during the IP with 5% BSA and 100ug/ml sheared salmon sperm DNA (Just make it up in IP buffer with inhibitors).

3) Use more stringent washes (increase salt concentration in later washes).

4) Try decreasing the amount of beads you use (although if you are using the magnetic beads this might not help very much).

Finally, for some proteins that are at low abundance on the chromatin or are difficult to pull down (thus difficult to see above background) we've had the most success with our 96 well plate based ChIP assay, Matrix ChIP (Nucleic Acids Research 2008 36: e17).

In any case, good luck and let me know if you have any other questions.

Joel



RE: blocking during the IP, I would dilute your chromatin in IP buffer with BSA and sssDNA before the antibody incubation. As far as the chelex goes, how much variability in volume are you getting. If it's +/- 5ul then you're probably ok (since this is going to be a small percentage, 2.5%, of your final volume of 200ul). If you are getting a large variability in volume (like 10ul or more) then you could probably weigh the stuff out and add it to each tube (about 10mg per tube). Sounds like a pain though.

-KPDE-

Yeah, I figured it wasn't a big deal, just wondering if people had tips, I'm getting more consistent.

-MKR-