- Add IP buffer according to your calculations. If the total vol-
ume of the IP should be 200μl, then the IP buffer should be
200 μl minus 20μl of agarose (50:50 slurry), minus 10μlof
antibody, minusxμl of lysate (Fig.1a)(seestep 8). - Add 10μl of antibody (seeNote 7).
- Addxμl of lysate. If you want to IP on 200μg of lysate and the
total protein concentration in that lysate is 5μg/μl, then add
40 μl of lysate (200μg divided by 5μg/μl) (seeNote 8). - Let the IPs rotate end over end at 4C overnight (seeNote 9).
- On Day 2, remove the IPs from rotation and spin down the
agarose for 1 min at 500gor quick spin in a small tabletop
centrifuge. Place tubes on ice. - Prepare a new set of tubes for the Post-IPs.
- Add 34μlof6SB to each of these tubes (seeNote 10).
- Transfer 170μl of the supernatant from the IP to the new tube
with 6SB and mix (Fig.1b). This is thePost-IP(seeNote
11 ). For the IP with irrelevant antibody, this is thePre-IP. - Wash the remaining agarose once in 180μl of IP buffer and
twice in 180μl of IP wash buffer. Between washes spin the
agarose down for 1 min at 500gand 4C or in a small
tabletop centrifuge. Use suction to discard the supernatant,
but be careful not to take away any agarose. Leave 10–20μl
of wash buffer to be safe. - After last wash, aspirate all supernatant, still being careful not
to lose any agarose beads. - Add 20μlof2SB to the agarose and mix gently without
splashing the agarose up the sides of the tube (Fig.1c). This
is the IP (seeNote 12). - Heat all samples (IPs and Pre- and Post-IPs) at 96C for 5 min.
Mix and spin down. - The samples are now ready for SDS-PAGE.
- The protein in the IP is no longer attached to the agarose but is
free in the small sample buffer supernatant. Do not load the
agarose on the gel but only the supernatant. - The resulting protein concentration in the Pre-IP and the Post-
IP is the same. The input was 200μg of lysate protein in 190μl
(half of the 20μl of agarose (50:50 slurry) was packed beads
and is not part of the supernatant,seestep 6). 170μl of the
supernatant was transferred to a new tube and diluted with
34 μlof6SB. This gives a concentration of: (200 μg/
190 μl * 170μl)/(170μlþ 34 μl)¼0.88μg/μl (Fig.1c)(see
Note 13).
208 Jesper B. Birk and Jørgen F. P. Wojtaszewski