Prebind MBD2a-Fc to Protein A Magnetic Beads (NEB #E2600)
Before proceeding, determine the number of samples to be analyzed. As a general rule of thumb, use 1 μl of MBD2-Fc protein and 10 μl Protein A Magnetic Beads for up to 10 μg of input DNA. For larger amounts of input DNA, scale the reaction accordingly (e.g., for an experiment using 100 μg of input DNA, add 10 μl MBD2a-Fc protein and 100 μl Protein A Magnetic Beads).
Resuspend Protein A Magnetic Beads by gently pipetting the slurry up and down until suspension is homogeneous. Alternatively, rotate the tube gently for 30 minutes at room temperature.
Prepare 1X Bind/Wash Buffer by diluting 1 part of 5X Bind/Wash Buffer with 4 parts of DNase-free water. One individual reaction (for up to 10 μg of input DNA) from start to finish will require ~5 mls of 1X Bind/Wash Buffer.
- Add 11 µl of 1X Bind/Wash Buffer, 10 µl of Protein A Magnetic beads and
1 µl of MBD2-Fc in one tube. Mix by ﬂicking the tube.
- Mix the bead-protein mixture by placing the tube in a rotating mixer for 15
minutes at room temperature.
a. Add 1000 μl of 1X Bind/Wash Buffer to the tube to wash the beads.
b. Mix the beads on a rotating mixer for 3 minutes at room temperature.
c. Place the tube on the magnetic rack for 2–5 minutes to concentrate all of the beads on the inner wall of the tube.
d. Carefully remove the supernatant with a pipette without disturbing the beads. Discard the supernatant.
- Repeat Step 3 two more times. Make sure all the supernatant is removed.
- Remove the tube from the rack and add 16 μl of 1X Bind/Wash Reaction Buffer. Resuspended the beads by pipetting up and down.
- The MBD2-Fc/Protein A Magnetic Bead mixture is stable for up to 1 week at 4°C.