Preparation of Protein A (G) Sepharose (45 min)

49. Weigh 0.25 g of protein A (or G) Sepharose beads into a 14-ml polypropylene tube.

50. Add 10 ml of H₂O, and mix.

51. Centrifuge the tubes for 3 minutes at 1500g in a swing-out rotor, and discard the supernatant.

52. Repeat Steps 50 and 51 four times.

53. Add 1 ml of H₂O, and resuspend the beads.

54. Distribute 100-µl aliquots into 10 1.5-ml microcentrifuge tubes. Store these aliquots at 4°C.
These aliquots are used for the extraction of antibody-bound chromatin from ChIP experiments (starting in Step 55).

Extraction of Immunoprecipitated Chromatin with Protein A (G) Sepharose (6-7 h)

55. Add 50 µl of protein A (or G) Sepharose (from Step 54) to each tube (from Step 48).

56. Rotate the tubes at 20-30 rpm for 4 hours at 4°C.

57. Centrifuge the tubes at 1500g in a swing-out rotor for 3 minutes.

58. Transfer the supernatant to a 2-ml microcentrifuge tube. Store it at 4°C.
This fraction contains the chromatin that did not link to the antibody (i.e., the "unbound fraction").

59. Resuspend the Sepharose beads in 1 ml of washing buffer A.

60. Transfer the resuspended beads to a 15-ml Falcon tube.

61. Bring the total volume to 10 ml with washing buffer A. Mix briefly.

62. Centrifuge for 3 minutes at 1500g in a swing-out rotor at 4°C. Carefully discard the supernatant.

63. Resuspend the beads in 10 ml of washing buffer B. Briefly mix.

64. Centrifuge for 3 minutes at 1500g in a swing-out rotor at 4°C. Carefully discard the supernatant.

65. Resuspend the Sepharose beads in 10 ml of washing buffer C.

66. Centrifuge for 3 minutes at 1500g in a swing-out rotor at 4°C. Carefully discard the supernatant.

67. To elute the chromatin, resuspend the Sepharose beads in 500 µl of ChIP elution buffer, and transfer the sample to a 1.5-ml microcentrifuge tube.

68. Incubate the samples for ~30 minutes at room temperature on a rotating wheel at 20-30 rpm. After this incubation, centrifuge for 3 minutes at 1500g in a microcentrifuge at room temperature.

69. Carefully transfer the supernatant into a 2-ml microcentrifuge tube. Store it at 4°C.
This tube contains the chromatin eluted from the Sepharose beads (i.e., the "bound fraction").
See Troubleshooting.

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