Protocol Online logo
Top : Forum Archives: : DNA Methylation, Histone and Chromatin Study

solid support for ChIP washes - (May/22/2007 )

I am exploring alternatives to the standard protein A bead immunoprecipitations and have read a number of posts on this forum with interest.

In one post a while back, pcrman wrote the following:

"To minimize sample loss during ChIP washes, we now use Qiagen small spin column which comes with the Oligotex System. We load ChIP agarose beads to the small spin column, and do all the washes on the column."

I assume that the oligotex spin columns have a porous membrane that allows soluble material to pass through but traps the protein A/G beads. If so, it would indeed be an attractive alternative to the usual method of centrifuging the beads, removing the supernatant, washing, etc. especially with multiple samples (and/or smaller volumes of beads).

Has anyone else tried this? I haven't seen any published reports using this method.
pcrman, could you comment further or provide additional details?

-jb-bcm-

Hi jb-bcm,

yes it works great for us. you can buy the column from qiagen (Qiagen #79523). the wash steps are as follows

After o/n incubation with antibody:

1. add 60 micro liter of protein G agarose and rotate for 1 hr at 4oC
2. Pellet agarose by centrifugation for 1 min at 3000-5000 g and remove most of the supernatant
3. Resuspend beads in 0.4 ml of low salt wash buffer. Transfer beads to a “Small Spin Column (Qiagen #79523) hold on ice for 5 min with occasional mixing. Spin and discard spin-through
4. Wash beads with 0.4 ml of low salt wash buffer twice
5. Wash beads with 0.4 ml of high salt wash buffer twice
6. Wash beads with 0.4 ml of LiCl wash buffer twice
7. Wash beads with 0.4 ml of TE buffer 3 times

-pcrman-

pcrman,

Thanks for the quick response. I'll order some columns right away and give it a "spin". Are there publications that can be referenced?
A few other questions... The spin columns are placed in a collection tube, right? How exactly are the washes "mixed"? Do you also elute through the column, and if so, what elution buffer do you use? I assume that you then proceed to some type of DNA purification step (PCI? another spin column?).

-jb

-jb-bcm-

Yes the spin columns are placed in a collection tube with cap. You have to maually shake or rack the tube once a while.

We use the spin columns along with the ChiP kit from upstates. the following steps are to follow step 7 I previously posted:
(we use qiagen PCR purification columns for the final DNA cleanup.

8. Spin column so that they are dry, transfer the column to a fresh microfuge tube

9. Prepare fresh elution buffer (420 mg NaHCO3, 5 ml 10% SDS, add H2O to 50 ml)

10. Add 100 micro liter elution buffer to each column and let stand at room temperature for 15 min with occasional agitation. Spin and collect the spin-through.

11. Elute with another 100 micro liter elution buffer and combine the elutes

12. Add 8 micro liter of 5 M NaCl, 1 micro liter of RNase A (10 mg/ml), incubate at 65°C for at least 4 hrs. Include Input at this step.

13. Take out tubes and let them cool to RT. Add 4 micro liter of 0.5 M EDTA, 8 micro liter 1 M Tris-HCl and 1 micro liter Proteinase K (20 mg/ml) and incubate at 45°C for 1-2 hrs.

14. Purify DNA using Qiagen PCR purification kit (#28104)

15. Elute DNA in 30-50 micro liter EB buffer

-pcrman-

QUOTE (pcrman @ May 23 2007, 11:20 PM)
Yes the spin columns are placed in a collection tube with cap. You have to maually shake or rack the tube once a while.

We use the spin columns along with the ChiP kit from upstates. the following steps are to follow step 7 I previously posted:
(we use qiagen PCR purification columns for the final DNA cleanup.

8. Spin column so that they are dry, transfer the column to a fresh microfuge tube
...
15. Elute DNA in 30-50 micro liter EB buffer


Thanks a bunch, pcrman. The instructions are very clear and follow the Upstate kit protocol (which I'm also trying) quite closely with the exception of the spin columns. I can't wait to give it a try. The main drawback I can see is the added cost of the oligotex columns, but if it saves in time/energy it may well be worthwhile.

-jb

-jb-bcm-

QUOTE (pcrman @ May 23 2007, 10:42 AM)
Hi jb-bcm,

yes it works great for us. you can buy the column from qiagen (Qiagen #79523). the wash steps are as follows

After o/n incubation with antibody:

1. add 60 micro liter of protein G agarose and rotate for 1 hr at 4oC
2. Pellet agarose by centrifugation for 1 min at 3000-5000 g and remove most of the supernatant
3. Resuspend beads in 0.4 ml of low salt wash buffer. Transfer beads to a “Small Spin Column (Qiagen #79523) hold on ice for 5 min with occasional mixing. Spin and discard spin-through
4. Wash beads with 0.4 ml of low salt wash buffer twice
5. Wash beads with 0.4 ml of high salt wash buffer twice
6. Wash beads with 0.4 ml of LiCl wash buffer twice
7. Wash beads with 0.4 ml of TE buffer 3 times



just a quick question, how long and at what speed do you spin after each wash?

oh and do you think the columns from promega (A7211) would be appropriate? they are much cheaper, probably cos they are not RNAse free. and i have a heap of them on site smile.gif

-frozenlyse-

The purpose of using column is just to provide solid support for the agarose beads so you won't lose any during washes. The length and speed for spinning is such that you just get rid of the washing buffer, but don't let the bead over dry as you would do with washes without using columns. I don't know whether promega column wil do the same job, as I said what you need is a solid support for the beads.

-pcrman-

QUOTE (pcrman @ May 24 2007, 07:25 PM)
The purpose of using column is just to provide solid support for the agarose beads so you won't lose any during washes. The length and speed for spinning is such that you just get rid of the washing buffer, but don't let the bead over dry as you would do with washes without using columns. I don't know whether promega column wil do the same job, as I said what you need is a solid support for the beads.


Since high speed can damage the beads, I do not think high speed centrifuge is good. So, what speed do you think is best for beads and column?

-chip417-