Download ChIP Lysis Buffer High Salt: sc-45001 Material Safety Data Sheet

Santa Cruz Biotechnology, Inc.
Material Safety Data Sheet
ChIP Lysis Buffer High Salt: sc-45001
DESCRIPTION
ChIP Lysis Buffer High Salt can be used for Chromatin Immunoprecipitation assays using the protocol provided below.
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Harvest beads by centrifugations at 12,000 rpm for 20 seconds
and place tube in ice.
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Wash beads twice with 1 ml Lysis Buffer High Salt (sc-45001).
Product size: 300 ml.
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Wash pellet four times with Wash Buffer (sc-45002).
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Resuspend beads in 400 µl Elution Buffer (sc-45003).
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Reverse cross-links by incubating tube in a 67º C water bath,
mixing occasionally over two hours. Remove beads by
centrifugation and continue incubating supernatant at 67º C
overnight.
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Centrifuge for 3 minutes at 10,000 to remove any residual beads
and save supernatant.
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To isolate DNA, extract supernatant once with 500 µl
phenol/chloroform/isoamyl alcohol (25:24:1), vortex thoroughly
and separate phases by centrifuging tube for 3 minutes at 14,000
rpm.
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Save the aqueous phase, back extract the organic phase once with
100 µl 10 mM Tris, 1 mM EDTA, pH 8.1 (TE) and pool
aqueous phases.
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Extract pooled aqueous phase with 600 µl chloroform/isoamyl
alcohol.
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DNA may be concentrated by using commercially available kits.
PROTOCOL
NOTE: ChIP protocols vary widely. The following protocol
should be suitable for most experiments.
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Wash cells twice with PBS at room temperature, resuspending to
approximately 5x105 cells/ml (approximately 2x107 cells total).
Add formaldehyde to a final concentration of 1% and incubate at
room temperature for 10 minutes.
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Terminate cross-linking reactions by adding glycine to a final
concen-tration of 0.125 M.
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Pellet cells (2,000 RPM, 5 minutes) and wash once with ice cold
PBS.
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Resuspend cells in 6 ml Lysis Buffer (sc-45000) by mixing gently.
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Collect crude nuclear extract by microcentrifugation at 2,000
rpm, 5 minutes.
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Wash again with PBS. Pellet may be frozen or processing may be
continued as follows:
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Resuspend pellet in ~1.9 ml Lysis Buffer High Salt (sc-45001)
and transfer to 2 ml microcentrifuge tube for the sonication step.
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Sonication conditions should be optimized since results may vary
using different sonifiers. The following conditions were
established by using a Sonics VC130 with a 3 mm tip probe.
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Sonicate on ice at power output setting = 5–6, continuous mode,
4 times at 30 second intervals.
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Centrifuge extract for 15 minutes, 10,000 rpm at 4° C and save
super-natant (chromatin).
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Determine protein concentration of supernatant.
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For the IP step we recommend using 100-500 µg protein and
0.1–1 µl TransCruz reagent (0.2–2 µg).
NOTE: Investigators may wish to consider using the primary
antibody conjugated to sepharose or magnetic beads as an
alternative to using secondary immunoprecipitation reagents (e.g.,
Protein A-Agarose) as described here. Combining primary
antibodies directed to different epitopes of the same protein may
be advantageous in some cases.
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Preclear the chromatin solution by adding 50 µl Protein A/G
PLUS-Agarose (sc-2003) and incubate for 30 minutes at 4º C.
Centrifuge at full speed for 5 minutes at 4º C.
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Add primary antibody to the supernatant and incubate overnight
at 4° C.
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Add 50 µl Protein A/G PLUS-Agarose (sc-2003) and incubate for
2 hrs at 4º C.
WARNING
ChIP Lysis Buffer contains Nonidet(R) P 40 substitute
(CAS#9016-45-9). Follow standard lab procedures when using this
product. In case of skin contact, flush with water and remove
contaminated articles. In case of eye contact, flush with water. In
case of swallowing, induce vomiting and wash out mouth with
water. Always wear full length clothing, safety gloves, and safety
glasses in a well-ventilated area.
STORAGE
Store at 4° C.
DISPOSAL
Contact a licensed professional waste disposal service to dispose of
this material. Dissolve or mix the material with a combustible
solvent and burn in a chemical incenerator equipped with an
afterburner and scrubber. Observe all federal, state and local
environmental regulations.
FOR RESEARCH USE ONLY; NOT FOR DIAGNOSTIC
OR THERAPEUTIC USE.
The above information is believed to be correct but does not
purport to be all inclusive and shall be used only as a guide. Santa
Cruz Biotechnology shall not be held liable for any damage
resulting from handling or from contact with the product.
U.S. 1.800.457.3801 • 831.457.3800 • fax 831.457.3801 • Europe + 800 4573 8000 • 49 (0)6221 4503 0 • fax 49 (0)6221 4503 45 • www.scbt.com
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