100 mM Tris (pH 8.0), 500 mM LiCl, 1% NP-40 and 1% Deoxycholate
ChIP Wash Buffer can be used for Chromatin Immuno- precipitation assays using the protocol provided below.
NOTE: ChIP protocols vary widely. The following protocol should be suitable for most experiments.
•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.
•Terminate cross-linking reactions by adding glycine to a final concentration of 0.125 M.
•Pellet cells (2,000 RPM, 5 minutes) and wash once with ice cold PBS.
•Resuspend cells in 6 ml Lysis Buffer (sc-45000) by mixing gently.
•Collect crude nuclear extract by microcentrifugation at 2,000rpm, 5 minutes.
•Wash again with PBS. Pellet may be frozen or processing may be continued as follows:
•Resuspend pellet in ~1.9 ml Lysis Buffer High Salt (sc-45001) and transfer to 2 ml microcentrifuge tube for the sonication step.
•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.
•Sonicate on ice at power output setting = 5–6, continuous mode, 4 times at 30 second intervals.
•Centrifuge extract for 15 minutes, 10,000 rpm at 4° C and save super-natant (chromatin).
•Determine protein concentration of supernatant. •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.
•Preclear the chromatin solution by adding 50 μl Protein A/G PLUS-Agarose (sc-2003) and incubate for 30 minutes at 4° C. Centrifuge at full speed for 5 minutes at 4° C.
•Add primary antibody to the supernatant and incubate overnight at 4° C.
•Add 50 μl Protein A/G PLUS-Agarose (sc-2003) and incubate for 2 hrs at 4° C.
•Harvest beads by centrifugations at 12,000 rpm for 20 seconds and place tube in ice.
•Wash beads twice with 1 ml Lysis Buffer High Salt (sc-45001).
•Wash pellet four times with Wash Buffer (sc-45002).
•Resuspend beads in 400 μl Elution Buffer (sc-45003).
•Reverse cross-links by incubating tube in a 67° C water bath, mixing occasionally over two hours. Remove beads by centrifugation and continue incubating supernatant at 67° C overnight.
•Centrifuge for 3 minutes at 10,000 to remove any residual beads and save supernatant.
•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.
•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.
•Extract pooled aqueous phase with 600 μl chloroform/isoamyl alcohol.
•DNA may be concentrated by using commercially available kits.
Store at 4° C
For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.
Download SDS (MSDS)
Certificate of Analysis
Adobe Acrobat Reader is required to reliably view, print and comment on PDF documents
ChIP Wash Buffer
See how others have used ChIP Wash Buffer. Click on the entry to view the PubMed entry
To place an order using RMB or to ship to mainland China, please visit www.scbio.cn
Create a new account
Email address already exists, please enter a new valid email address.
USE YOUR SOCIAL NETWORK
Create an account quickly and easily with your preferred social network account. You won't have to remember an extra name and password.
Creating an account with us makes your shopping experience much easier and faster. You can save favorites, save cart, check order status and speed through checkout with saved addresses, payment methods and more.