Good thing takes time

Method 1

1. Add colcemid (1:1000 KaryoMAX) directly to culture dish and swirl.
Incubate 30 min to 2 hours.

• Metaphases can be prepared without colcemid. Colcemid should increase the
number of metaphase chomosomes but longer incubation times will result in shorter,
more compact chromosomes.

2. Trypsinize cells as normal and wash 1X 10ml PBS. At this point it is no longer
necessary to be sterile.

3. Remove as much PBS as possible and gently resuspend the cells in the residual.

4. Slowly add 0.075M KCl dropwise to 10ml. I add 1-2 drops then invert
the tube. As soon as there are about 3 ml of KCl in the tube addition can
become faster.

5. Incubate at 37oC (in a water bath) for EXACTLY 6 minutes.

6. Centrifuge at 900rpm for 5 minutes.

7. Remove as much KCl as possible and gently resuspend the cells in the residual.

8. SLOWLY add 5 ml of fixative (3:1 Methanol/Acetic acid; prepared fresh)
dropwise and carefully mix the whole time. Adding fixative too quickly will
result in clumping.

9. Centrifuge at 900rpm for 5 minutes and remove fixative

10. Slowly add 2 ml fixative dropwise.

11. Centrifuge 900rpm 5 minutes and remove all but 200-500μl of the fixative.
Cells are stable for extended times in fixative. If desired, store at 4oC.

12. Drop a few drops from about 18 inches high onto angled, humidified
microscope slide.

13. IMMEDIATELY blow on the slide very gently.

14. Air dry at least 10 minutes. Slides are now stable for a long time.



Method II

1. Add colcemid (1:1000 KaryoMAX) directly to culture dish and swirl.
Incubate 30 min to 2 hours.
• Metaphases can be prepared without colcemid. Colcemid should increase the
number of metaphase chomosomes but longer incubation times will result in shorter,
more compact chromosomes.

2. Trypsinize cells as normal and wash 1X 10ml PBS. At this point it is no longer
necessary to be sterile.

3. Remove as much PBS as possible and gently resuspend the cells in the
residual.

4. Slowly add KCl dropwise to 10ml. I add 1-2 drops then invert
the tube. As soon as there are about 3 ml of KCl in the tube addition can
become faster.
use 0.4% KCl for MEFs
use 0.57% KCl for lymphocytes, ES cells

5. Incubate at 37oC (in a water bath) for EXACTLY 8 minutes.

6. Centrifuge at 900rpm for 5 minutes.

7. Remove as much KCl as possible

8. Gently add 10 ml of cold fixative (3:1 methanol/acetic acid prepared fresh) and
gently but quickly resuspend the pellet by pipetting up and down.

9. Centrifuge at 900rpm for 5 minutes

10. Repeat steps 8 and 9

11. Remove all but 1-2 ml of fixative and gently resuspend pellet in remainder

12. Store at 4oC or proceed to dropping metaphases onto slides.



Method III
(cultured lymphocytes cells)
1. Add KaryoMax colcemid to culture at a 1:200 dilution. Incubate 3.5-4.5 hours.

2. Spin cells down in tabletop clinical centrifuge for 5 min at 1000rpm in 15ml
concial tubes.

3. Remove supernatant and gently resuspend in 1ml PBS pH 7.4 by pipetting up and
down with a P1000.

4. Spin cells down in microfuge for 2min at 1000 RCF.

5. Very gently remove most of the supernatant with a P1000, being careful not to
disturb the pellet. Gently resuspend in the residual PBS by flicking the tube or
pipetting up and down with a p200.

6. Add 900μl 0.075M KCl. Incubate at 37oC for 17 min.

7. Slowly and gently add 100μl cold, fresh fixative (3:1 methanol/acetic acid).
Invert to mix.

8. Spin down in a microfuge for 2min. at 1500 RCF.

9. Very gently remove most of the supernatant with a P1000, being careful not to
disturb the pellet. Gently resuspend in the residual fixative by flicking the tube.

10. Add 1ml cold fixative. Invert tube

11. Spin down in a microfuge for 2min. at 1500 RCF

12. Very gently remove most of the supernatant with a P1000, being careful not to
disturb the pellet. Gently resuspend in the residual fixative by flicking the tube.

13. Add 1ml cold fixative. Invert tube

14. Spin down in a microfuge for 2min. at 1500 RCF

15. Remove all but 50-100μl of fixative, and gently resuspend cells in the remainder.

16. Store at -20oC.

The procedure is cited from the website http://www.abgent.com/doc/if1


==============================================================================

Step 1: Rinse cells attached to cover slips twice with PBS, removing liquid by gentle aspiration in this and subsequent steps.

Step 2: Fixation permeabilization:

Option I:

* Fix cells with 4% formaldehyde (in some paper they also use 2%) in PBS for 6 min at room temperature, then rinse briefly twice with PBS**.
* Permeabilize fixed cells with 0.2% Triton X-100 (in some paper they use 0.5%, and that is what I did) in PBS for 6 min.

Option II:

* Fix/permeabilize cells in -20C methanol for 6 minutes**.


[**Note: At this stage, the procedure may be interrupted for long term storage. Coverslips immersed in PBS and stored at 4C are good for several weeks.]

Step 3: Wash cells briefly 3 times with PBS, then 2 times with PBS containing 5% BSA (blocking reagent).

Step 4: Dilute primary antibody in PBS/5% BSA. Working quickly, aspirate area surrounding coverslip to dryness, then gently add 100 ml of diluted primary antibody to the coverslip, so that solution remains restricted to coverslip by surface tension. Incubate for 1 hour at room temperature in a moist environment to prevent drying.

Step 5: Wash cells 3 times with PBS, then 2 times with PBS/5% BSA.

Step 6: Dilute fluorochrome-coupled secondary antibody in PBS/5% BSA and apply as in step 5. Incubate 1 hr. at room temperature.

Step 7: Wash cells 3 times with PBS, then mount coverslips to slides using antifade mounting medium.


Considerations:

* Adherent cells may be grown directly on coverslips or chambered slides; suspension cells may be adhered to coverslips via poly-L-lysine treatment.

* Care should be taken to use the highest quality primary and secondary antibodies in order to avoid non-specific labeling. Ideally the specificity of primary antibodies is confirmed via immunoblotting of cell extracts. A control immunofluorescence sample omitting the use of primary antibody will reveal the extent of non-specific signal generated by the secondary antibody.

* In case of high background, the use of less primary and/or secondary antibodies as well as increased or alternative blocking reagent can be considered. 10% serum in PBS is another useful blocking agent - use only serum that will not cross-react with secondary antibody.

* If the assay involves localization of a protein expressed from a heterologous promoter, then the researcher should keep in mind that overexpression of the protein may produce mislocalization and hence broader staining than expected from endogenous expression.

* Several approaches can be considered in cases of an unacceptably low signal. The immunofluorescence protocol itself may be altered: use increased amounts of primary antibody, extend the incubation of primary antibody to overnight at 4C, or use a differnt fixation/permeabilization regimen (gluteraldehyde, acetone, others).

Representative blot from a previous lot.
Anti-acetylated Histone H3 immunoprecipitated
chromatin from lysates of
quiescent 3T3/A31 cells (lane 1) or
sodium butyrate treated 3T3/A31 cells
(lane 2). The immunoprecipitate was
resolved by electrophoresis, transferred
to nitrocellulose and probed with antiacetyl
Histone H3 (0.2 μg/mL, Catalog #
06-599). Proteins were visualized using
a goat anti-rabbit secondary antibody
conjugated to HRP and a chemiluminescence
detection system. Arrow
indicates acetylated Histone H3
(17kDa).

Quality Control Testing
Chromatin Immunoprecipitation: 5-10 μL of antiacetyl Histone H3 polyclonal antibody (Catalog # 06-599) immunoprecipitated acetylated histone H3 as determined by chromatin immunoprecipitation using actively growing HeLa cells and subsequent end
point PCR using primers specific to the GAPDH promoter (Catalog #22-004).
Chromatin Immunoprecipitation may also be performed using 10 μL of anti-acetyl Histone H4 polyclonal antibody (Catalog # 06-866) to immunoprecipitate acetylated histone H4.



Reagent:

Protein A Agarose/Salmon Sperm DNA, Catalog #
16-157C. One vial containing 1.5 mL packed beads
with 600 μg sonicated salmon sperm DNA, 1.5 mg BSA
and approximately 4.5 mg recombinant Protein A.
Provided as a 50% gel slurry for a final volume of 3 mL
per vial. Suspended in TE buffer, pH 8.0, containing
0.05% sodium azide. Liquid suspension.

ChIP Dilution Buffer, Catalog # 20-153. Two vials,
each containing 24 mL of 0.01% SDS, 1.1% Triton X-
100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM
NaCl.

Low Salt Immune Complex Wash Buffer, Catalog #
20-154. One vial containing 24 mL of 0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1,
150 mM NaCl.

High Salt Immune Complex Wash Buffer, Catalog #
20-155. One vial containing 24 mL of 0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1,
500 mM NaCl.

LiCl Immune Complex Wash Buffer, Catalog #
20-156. One vial containing 24 mL of 0.25 M LiCl,
1% IGEPAL-CA630, 1% deoxycholic acid (sodium
salt), 1 mM EDTA, 10 mM Tris, pH 8.1.

TE Buffer, Catalog # 20-157. Two vials, each
containing 24 mL of 10 mM Tris-HCl, 1 mM EDTA,
pH 8.0.

0.5M EDTA, Catalog # 20-158. One vial
containing 250 μL of 0.5 M EDTA, pH 8.0.
5M NaCl, Catalog # 20-159. One vial containing
500 μL of 5 M NaCl.

1M Tris-HCl, pH 6.5, Catalog # 20-160. One vial
containing 500 μL of 1 M Tris-HCl, pH 6.5.
SDS Lysis Buffer, Catalog # 20-163. One vial
containing 10 mL of 1% SDS, 10 mM EDTA, 50
mM Tris, pH 8.1.



Application References
1. Sundaraj, K.P., et al., J. Biol. Chem. 279: 6152-6162, 2004.
2. Detich, N., et al., J. Biol. Chem. 278: 27586-27592, 2003.
3. Gummow, B.M., et al., J. Biol. Chem. 278: 26572-26579, 2003.
4. Cervoni, N., & Szyf, M., J. Biol.Chem. 276: 40778-40787, 2001.
5. Manabe, I., et al., J. Clin. Invest. 107: 823-834, 2001.
General References:
6. Luo, R.X., et al., Cell 92: 463-473, 1998.
7. Braunstein, M., et al., Mol. Cell. Biol. 16: 4349-4356, 1996.



Other components required but not included as part of kit are:
Reagents Equipment
• Cells, stimulated or treated as appropriate for the
experimental system
• Antibody for chromatin immunoprecipitation
• 37% Formaldehyde
• PBS
• Dry ice
• Elution buffer: 1% SDS, 0.1 M NaHCO3
• Molecular Biology grade Proteinase K
• Glycogen or tRNA
• 50% Phenol/50%Chloroform (containing 1% isoamyl alcohol)
• 95% Ethanol
• 70% Ethanol
• vortex mixer
• rotating wheel/platform
• shaking incubator
• timer
• variable volume (5-1000 μL) pipettes and




Chromatin Immunoprecipitation Protocol
Part A. Optimization of DNA Shearing
Establish optimal conditions required for shearing cross-linked DNA to 200-1000 base pairs in length by following
steps 1- 9 below. Vary the power setting and/or the number of 10-second pulses during sonication of the samples.
Be sure to keep the sample on ice at all times (the sonication generates heat which will denature the DNA). Check
the size of sonicated DNA by gel electrophoresis after reversion of cross-links. Our experience shows DNA is
sheared to the appropriate length with 3-4 sets of 10-second pulses using a Cole Parmer, High Intensity Ultrasonic
Processor/Sonicator, 50-watt model equipped with a 2 mm tip and set to 30% of maximum power. Once
sonication conditions have been optimized, keep cell number consistent for subsequent experiments. The
protocol below for the optimization of DNA Shearing is for one Chip assay (~1 x 106 cells per condition).
Note: Steps 3-7 should be done on ice.

1. Stimulate or treat 1 x 106 cells on a 10 cm dish as appropriate. (Cells should be treated under conditions for
which transcriptional activation of the gene of interest has been demonstrated). Include one extra dish (1 X 106
cells) to be used solely for estimation of cell number.
2. Cross link histones to DNA by adding formaldehyde directly to culture medium to a final concentration of 1%
and incubate for 10 minutes at 37°C. (For example, add 270 μL 37% formaldehyde into 10 mL of growth
medium on plate).
3. Aspirate medium, removing as much medium as possible. Wash cells twice using ice cold PBS containing
protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL aprotinin and 1 μg/mL pepstatin A).
Note: Add protease inhibitors to PBS just prior to use. PMSF has a half-life of approximately 30 minutes in
aqueous solutions.
4. Scrape cells into conical tube.
5. Pellet cells for 4 minutes at 2000 rpm at 4°C. Warm SDS Lysis Buffer (Catalog # 20-163) to room temperature
to dissolve precipitated SDS and add protease inhibitors (inhibitors: 1mM PMSF, 1 μg/mL aprotinin and 1
μg/mL pepstatin A).
6. Resuspend cell pellet in 200 μL of SDS Lysis Buffer (Catalog # 20-163) and incubate for 10 minutes on ice.
Note: The 200 μL of SDS Lysis Buffer is per 1 X 106 cells; if more cells are used, the resuspended cell pellet
should be divided into 200 μL aliquots so that each 200 μL aliquot contains ~1 X 106 cells.
7. Sonicate lysate to shear DNA to lengths between 200 and 1000 basepairs being sure to keep samples ice cold
(Note: Once sonication conditions have been optimized following steps 1 to 9, proceed to Part B, step 1
below).
8. Add 8 μL 5 M NaCl (Catalog # 20-159) and reverse crosslinks by heating at 65°C for 4 hours.
9. Recover DNA by phenol/chloroform extraction and run sample (example 5 μL, 10 μL and 20 μL samples) in an agarose gel to visualize shearing efficiency.



Part B. Experimental protocol.
If sonication conditions have been optimized (Part A), complete steps 1 through 7 below and and continue with the protocol that follows. For a negative/background control, prepare a sample to use as a no-antibody immunoprecipitation control in step 5 below. Additionally, transcriptionally unactivated DNA samples should be prepared as controls for PCR in section II.

1. Centrifuge samples (from part A, step 7) for 10 minutes at 13,000 rpm at 4°C, and transfer the supernatant to a
new 2 mL-microcentrifuge tube. Discard pellet.
2. Dilute the sonicated cell supernatant 10 fold in ChIP Dilution Buffer (Catalog # 20-153), adding protease inhibitors as above. This is done by adding 1800 μL ChIP Dilution Buffer to the 200 μL sonicated cell supernatant for a final volume of 2 mL in each immunoprecipitation condition. Note: If proceeding to PCR a portion of the diluted cell supernatant 1% (~20 μL) can be kept to quantitate the amount of DNA present in different samples at the PCR protocol, Part B, section II, step 6. This sample is considered to be your input/starting material material and needs to have the Histone-DNA crosslinks reversed by adding 1 μL of 5 M NaCl and heating at 65°C for 4 hours (see section II, step 3).

3. To reduce nonspecific background, pre-clear the 2 mL diluted cell supernatant with 75 μL of Protein A
Agarose/Salmon Sperm DNA (50% Slurry), (Catalog # 16-157C) for 30 minutes at 4°C with agitation.
4. Pellet agarose by brief centrifugation and collect the supernatant fraction.
5. Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2 mL supernatant fraction and incubate overnight at 4°C with rotation. For a negative control, perform a no-antibody immunoprecipitation by incubating the supernatant fraction with 60 μL of Protein A Agarose/Salmon Sperm DNA (50% Slurry), (Catalog # 16-157C) for one hour at 4°C with rotation and proceed to step 7.
6. Add 60 μL of Protein A Agarose/Salmon Sperm DNA (50% Slurry), (Catalog # 16-157C) for one hour at 4°C
with rotation to collect the antibody/histone complex.
7. Pellet agarose by gentle centrifugation (700 to 1000 rpm at 4°C, ~1min). Carefully remove the supernatant that
contains unbound, non-specific DNA. Wash the protein A agarose/antibody/histone complex for 3-5 minutes on
a rotating platform with 1 mL of each of the buffers listed in the order as given below:
a) Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one wash
b) High Salt Immune Complex Wash Buffer (Catalog # 20-155), one wash
c) LiCl Immune Complex Wash Buffer (Catalog # 20-156), one wash
d) TE Buffer (Catalog # 20-157), two washes

After step 7 above, the sample is now a protein A/antibody/histone/DNA complex ready for either an IP/Immunoblot assay (Section I) or Polymerase Chain Reaction (PCR) assay (Section II):

Section I. Immunoprecipitation/Immunoblot protocol to detect histone.
1. Following washing of the beads in part B, step 7, immunoprecipitated histones can be analyzed by immunoblot
analysis. Add 25 μL of 1X Laemmli buffer per sample and boil for 10 minutes. Load 20 μL per lane and
perform immunoblot procedure as described per appropriate antibody.

Section II. PCR protocol to amplify DNA that is bound to the immunoprecipitated histone.
1. Freshly prepare elution buffer (1%SDS, 0.1M NaHCO3).
2. Elute the histone complex from the antibody by adding 250 μL elution buffer to the pelleted protein A
agarose/antibody/histone complex from step 7d above. Vortex briefly to mix and incubate at room temperature
for 15 minutes with rotation. Spin down agarose, and carefully transfer the supernatant fraction (eluate) to
another tube and repeat elution. Combine eluates (total volume = ~500 μL).
3. Add 20 μL 5 M NaCl (Catalog # 20-159) to the combined eluates (500 μL) and reverse histone-DNA crosslinks
by heating at 65°C for 4 hours. At this step the sample can be stored and –20°C and the protocol continued
the next day.
Note: Include the input/starting material (the sample saved from Part B, step 2, which has had the Histone-DNA
crosslinks reversed by adding 1 μL of 5 M NaCl per 20 μL sample and heating to 65°C for 4 hours) as well as a
transcriptionally-unactivated DNA sample as negative and background controls for the PCR reaction.
Previously, a 5 μL sample has been used in a nested PCR reaction. However, the amount of sample used per
reaction must be determined empirically (e.g., titrate the sample at this step by using 1, 2, 5, or 10 μL per PCR
reaction). If PCR results are poor, complete steps 4, 5 and 6 below to purify the DNA sample. NOTE: Handle
the samples carefully; some DNA may be lost during the purification steps.
4. Add 10 μL of 0.5 M EDTA (Catalog # 20-158), 20 μL 1 M Tris-HCl, pH 6.5 (Catalog # 20-160) and 2 μL of 10
mg/mL Proteinase K to the combined eluates and incubate for one hour at 45°C.
5. Recover DNA by phenol/chloroform extraction and ethanol precipitation. Addition of an inert carrier, such as 20
μg glycogen or yeast tRNA, helps visualize the DNA pellet. Wash pellets with 70% ethanol and air dry.
6. Resuspend pellets in an appropriate buffer for PCR or slot-blot reactions. PCR or slot-blot conditions must be
determined empirically.