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Home > Molecular Biology > Protein > Immunoprecipitation > ChIP

ChIP-Seq with M2-FLAG Antibody

This protocol is adapted from Aparicio, O. et al (2005) Curr Protoc Mol Biol. Last modified on March 21, 2010.

I have modified the original ChIP-Seq protocol to make it is suitable for Micrococcal Nuclease (MNase) digested chromatin. In addition, I have modified the yeast lysis buffer and MNase digestion buffer. Thus, cell lysis, MNase digestion, and chromatin IP could be done in the same buffer with minimum modifications. Anti-HA version of this Chip-Seq protocol can be found here: ChIP Seq with HA Antibody.

I. Cross-link protein-DNA complexes in vivo

  1. Grow 50 ml yeast culture overnight. Next morning inoculate 1 liter YPD, bring cell culture to 1x10^6 cells per ml (OD600 0.1 Abs). Grow the culture until mid log phase.
  2. Incubate cells with 1% formaldehyde for 20 min at room temperature (use 37% stock).
  3. Stop cross-linking with 0.125 M heat-sterilized Glycine, incubate another 5 minutes.

II. Harvest cells

  1. Collect cells by filtering cell culture through a 0.45 micron Nylon membrane filter. Vortex the filter membrane in ice-cold TBS to collect the cells.
    10 liters cell culture at OD600=1.0 Abs yields 10 g cell pellet.
  2. Centrifuge cells 5 min at 2500 x g, 4°C. Discard supernatant into a chemical waste container and resuspend pellet in 50 ml ice-cold TBS. Repeat once.
    Important: If you want to divide the cell pellet into equal aliquots, do so before washing with FA-M2 Lysis buffer (next step), because it is difficult to divide the pellet in FA-M2 Lysis buffer since it foams due to detergent.
  3. Resuspend cells in 10 ml ice-cold FA-M2 Lysis buffer.
  4. Pellet cells by centrifuging in a benchtop centrifuge 5 min at 3,000 x g, 4°C. Discard supernatant.
    The cells can remain on ice for a few hours while other samples are collected. Alternatively, the cells may be frozen in liquid nitrogen for storage in -80°C.

III. Lyse cells and istolate chromatin

  1. Resuspend the cell pellet in 1 ml ice-cold M2 FA lysis buffer/2 mM PMSF. Fill threequarters of a 2-ml flat-bottomed screw-cap microcentrifuge tube with 0.5-mm diameter silica-zirconia or glass beads. Add cells, taking care to avoid introduction of bubbles, and screw the cap on tightly. Make sure there are no leaks.
    The mini bead beater is recommended, because it is more efficient at breaking cells (multiple samples can be broken simultaneously). Silica-zirconia beads are more efficient at breaking cells than glass beads and are also recommended. To facilitate cell breakage with the mini bead beater, it is important that the final suspension nearly fill the tube. Do not break >160 OD600 units of cells (i.e., <5 x 10^9 cells) in a single 2-ml tube; for larger cultures, split the cells into multiple tubes.
  2. Lyse cells 3 min with a mini bead beater (MBB) at maximum speed. Remove sample and incubate 1 min in an ice-water bath. Repeat four times for a total breakage time of 15 min. If you are using bead beater (BB), 6 minutes lysing is enough. However, you must stop after every 1 min beating and keep the cells on ice at least 2-minutes. If you want to lyse your cells for 9 minutes, you really need to make sure that sample is still cold, after 6 minutes samples really get hot.
    This step assumes breakage with silica-zirconia beads. The cell breakage time with glass beads may be longer.

IV. Isolate Lysate

  1. Cut a 5-ml syringe 1cm below the flared opening (i.e., where the plunger is inserted) with a razor. Insert the smaller portion into a 15-ml disposable conical tube so that the flared portion of the truncated syringe rests on top of the conical tube opening, forming a microcentrifuge-tube holder.
  2. Invert the sample tube and punch a hole in the bottom with a 22-G or 25-G needle. Place the sample tube into the syringe/conical tube and punch a hole in the top cover with the same needle.
  3. Spin the assembly in a benchtop centrifuge 1 min at 1000 rpm, 4°C. Place the conical tube on ice. Discard the 2-ml centrifuge tube containing the dry beads after confirming the sample has been transferred to the 15-ml tube.
    Occasionally, beads will clog the pierced hole and prevent complete transfer of the sample. If this occurs, pierce the tube one or two more times and repeat the step in the same 15-ml tube. No additional buffer should be added.

V. MNase digest DNA

  1. Transfer the sample to a standard 1.5-ml microcentrifuge tube. Microcentrifuge 15 min at maximum speed (21,000 x g), 4°C. Discard the supernatant and resuspend the pellet with ice-cold ***MNase reaction buffer***. Add enough buffer that would give 0.3 g cell pellet/ml concentration. For example, if your starting material was 3 g yeast cell pellet, resuspend the chromatin in 10 ml MNase reaction buffer. If you make it more concentrated, MNase digestion does not look very good. I believe this is because chromatin is not very well dissolved at higher concentrations.
    The pellet contains the cross-linked chromatin, cell debris, and unbroken cells. The purpose of this centrifugation step is to remove soluble protein, most of which is not crosslinked to DNA, as it might contribute to nonspecific background in the subsequent immunoprecipitations step.
  2. Digest cross-linked chromatin by MNase. Stop digestion by adding EDTA at a concentration of 10 mM.
    First, test digest a faction of cross-linked chromatin by MNase to find the best digestion conditions. Analyze it as described below (steps i, ii, and iii).
  3. Centrifuge the digested chromatin at ~13,000 x g for 20 minutes, 4°C.
    Upon MNase digestion, the cross-linked chromatin is solubilized and purified away from the pelleted material which contains cell debris and unbroken cells. The resulting chromatin solution constitutes the input sample for the subsequent immunoprecipitation. The frozen aliquots are stable for many months when stored at -80°C and are suitable for immunoprecipitations.

VI. Check MNase digested chromatin (Test digest)

  1. For each time point of digestion, do following:
    Add equal volume of ChIP elution buffer to 250 microliter MNase digested chromatin aliquot.
    Add Pronase from 20 mg/ml stock solution at a concentration of 0.8 mg/ml (20 microliter for 500 microliter sample volume).
    Incubate 2 hr at 42°C, followed by 6 hr at 65°C.
  2. Extract with Phenol/Chloroform, and then with Chloroform (1x Vol).
    No need for ethanol precipitation here!
    Add RNAase A (20 microg/ml) and incubate for half an hour or longer at 37°C. Extract with Phenol/Chloroform, and with Chloroform alone. Add 1/10 V of 3 M NaAcetate pH 5.2 and glycogen (optional) to precipitate.
  3. Run half of the DNA on an agarose gel to check MNase digested DNA.

VII. Immunoprecipitation

  1. Save the supernatant from step-15; bring Tris.Cl concentration to 50 mM, and NaCl concentration to 150 mM (to the levels in lysis buffer) Also, add EDTA and Triton X-100.
    Save some lysate (input) for Western blotting to check the efficacy of pull down later.
  2. Take enough resin, and equilibrate it with TBS buffer.
  3. Add the M2 resin to the protein extract.
  4. Incubate Anti-Flag M2 affinity gel for about 90 minutes by mixing at room temperature.
  5. Collect the resin by centrifugation at 1000 x g for 5 min.
  6. Wash twice with TBS-T (Tween-20 at 0.05%), and wash four times with TBS at 4°C.
    Save 10 percent of beads for Western blotting.

VIII. Elution of Flag-fusion protein with 3X FLAG peptide

  1. Prepare 3X FLAG elution solution:
    Dissolve 3X FLAG peptide (Product Code F4799) in 0.5 M Tris HCl, pH 7.5, with 1 M NaCl at a concentration of 25 microg/microl. Dilute 5-fold with water to prepare a 3X FLAG stock solution containing 5 microg/microl of 3X FLAG peptide.
    For elution, add 3 microliters of 5 microg/microl 3X FLAG peptide stock solution to 100 microl of TBS (150 ng/microliter final concentration suggested by Sigma).
  2. Add enough (to 1 ml) of 3X FLAG elution solution to each sample and control resin (3x FLAG-peptide at 225 micrograms/ml).
  3. Incubate the samples and controls with gentle shaking for 30 min-2 hours at 2-8°C.
  4. Centrifuge the resin for 30 seconds at 5,000-8,200 x g. Transfer the supernatants to fresh test tubes. Be careful not to transfer any resin.
    Save all the resin, elute with SDS loading buffer, use for western blotting to test the efficacy of specific peptide elution.
  5. For immediate use, store the supernatants at 2-8°C. Store at -20°C for long term storage.

IX. Reverse cross-links and purify DNA

  1. Reverse cross-links and purify DNA as described at Paragraph VI, steps i-iii.

    2. Reagents and Solutions

    FA-M2 Lysis Buffer for ChIP with M2-Flag Antibody

    50 mM Tris.HCl pH 7.5
    150 mM NaCl
    1 mM EDTA
    1% Triton X-100

    Recipe for 1 liter FA-M2 Lysis Buffer:

    1M Tris.HCl pH 7.5  50 ml 
    5M NaCl 30 ml
    0.5M EDTA 2 ml
    Triton X-100 10 ml

    Mix the reagents above and adjust the volume to 1 liter with ultra-pure water. Store up to 1 year at room temperature. Freshly add 2 mM (f.c.) PMSF before use.

    Note: DO NOT use "SDS" and "Dexoycholate" in FA Lysis buffer since they will denature M2-FLAG antibody and inhibit M2 binding to FLAG fusion proteins!

    MNase reaction buffer

    10 mM Tris.Cl pH 7.5
    10 mM NaCl
    3 mM MgCl2
    2 mM CaCl2
    0.1 (v/v) NP-40

    Store buffer with above components up to 1 year at room temperature. Immediately before use, add 1 mM PMSF (fc) (from stock in ethanol).

    Note: Do not use more than 0.1% NP-40 (IGEPAL-630) in MNase reaction/IP buffer. This will inhibit binding of M2-FLAG antibody to FLAG fusion proteins!

    ChIP elution buffer

    50 mM Tris.Cl pH 7.5
    10 mM EDTA
    1% SDS

    Store up to 1 year at room temperature.