Chromatin Immunoprecipitation: Difference between revisions

Line 74:

==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====

# Add 20 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.

# Add 20 μl re-suspended magnetic bead slurry (for each sample you plan to use with this antibody) to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Vortex briefly to mix well.

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

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

# Repeat Steps b and c 3 times.

# Add 200 μl PBS/BSA to beads.

# Add 200 μl PBS/BSA (for each sample you plan to use this antibody with) to beads.

# Add 1 μg primary antibody. Do not vortex beads after adding the antibody.

# Add 1 μg primary antibody. Mix gently by tapping--Do not vortex beads after adding the antibody.

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

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

# Resuspend in 100 μl PBS/BSA, and proceed to Step 2.

# Resuspend in 100 μl PBS/BSA (for each sample you plan to use this antibody with), and proceed to Step 2.

==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====

@@ Line 74: / Line 74: @@
 ==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
-# Add 20 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.
+# Add 20 μl re-suspended magnetic bead slurry (for each sample you plan to use with this antibody)  to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Vortex briefly to mix well.
 # Place the microfuge tubes on the magnet rack and remove supernatants.
 # Resuspend the beads in 1 ml cold PBS/BSA.
 # Repeat Steps b and c 3 times.
-# Add 200 μl PBS/BSA to beads.
+# Add 200 μl PBS/BSA (for each sample you plan to use this antibody with) to beads.
-# Add 1 μg primary antibody. Do not vortex beads after adding the antibody.
+# Add 1 μg primary antibody. Mix gently by tapping--Do not vortex beads after adding the antibody.
 # Gently mix on a rotator platform for at least 2 hours at 4°C.
 # Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
-# Resuspend in 100 μl PBS/BSA, and proceed to Step 2.
+# Resuspend in 100 μl  PBS/BSA (for each sample you plan to use this antibody with), and proceed to Step 2.
 ==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====