Chromatin Immunoprecipitation: Difference between revisions

Line 65:

===Immunoprecipitation===

''Perform all steps in an ice bucket or in the cold room at 4°C.''

1. Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads:

<nowiki> a. Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold

PBS/BSA. Vortex briefly to mix well.

b. Place the microfuge tubes on the magnet rack and remove supernatants.

c. Resuspend the beads in 1 ml cold PBS/BSA.

d. Repeat Steps b and c 3 times.

e. Add 200 μl PBS/BSA to beads.

f. Add 5 μg primary antibody. Do not vortex beads after adding the antibody.

g. Gently mix on a rotator platform for at least 2 hours at 4°C.

h. Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.

i. Resuspend in 100 μl PBS/BSA, and proceed to Step 2.</nowiki>

2. Incubate bead-antibody complex with fragmented, cross-linked chromatin

<nowiki> a. Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from

Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at

4°C.

b. Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.

c. Remove and discard supernatant.

d. Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.

e. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.

f. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.</nowiki>

3. Reverse cross-linking and recover

===Analysis of Immunoprecipitated DNA===

@@ Line 65: / Line 65: @@
 ===Immunoprecipitation===
+''Perform all steps in an ice bucket or in the cold room at 4°C.''
+. Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads:
+   <nowiki> a. Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold
+       PBS/BSA. Vortex briefly to mix well.
+   b. Place the microfuge tubes on the magnet rack and remove supernatants.
+   c. Resuspend the beads in 1 ml cold PBS/BSA.
+   d. Repeat Steps b and c 3 times.
+   e. Add 200 μl PBS/BSA to beads.
+   f. Add 5 μg primary antibody. Do not vortex beads after adding the antibody.
+   g. Gently mix on a rotator platform for at least 2 hours at 4°C.
+   h. Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
+   i. Resuspend in 100 μl PBS/BSA, and proceed to Step 2.</nowiki>
+. Incubate bead-antibody complex with fragmented, cross-linked chromatin
+   <nowiki> a. Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from
+      Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at
+°C.
+   b. Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.
+   c. Remove and discard supernatant.
+   d. Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.
+   e. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.
+   f. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.</nowiki>
+. Reverse cross-linking and recover
 ===Analysis of Immunoprecipitated DNA===