Co-Immunoprecipitation
NHEK were cultured in T75-Cell flasks, washed 1x with PBS +10 g/l EDTA
and 2x with PBS. 1 ml Ca2+-free-PBS with ½ NaCl
concentration +1 % triton-X100 +1 % Nonoxinol-40 +0.1 %
sodium-dodecyl-sulfate +cOmplete™ (Merk, USA) was used for cell lysis
shaking 15 min on ice. Buffer without SDS, 0.5 % Triton-X100 and 0.1 %
Nonoxinol-40 was used for washing. The cells were mechanically detached
and sheared with a 5 ml syringe and 10G needle (B.Braun, Germany) 10x.
The resulting suspension was centrifuged at 4 °C for 15 min at 13.000
rpm and the pellet was removed. The protein amount was determined with a
commercial Pierce BCA protein assay kit. The supernatant was added to
buffer-washed Agarose G beads (Milipore, USA) adding about 600-1000
µg/protein. After 1.5 h on a rotator, the beads were removed via
centrifuging for 2 min at 4 °C, 8.000 rpm and the supernatant was mixed
with 1.5 µl of antibody or normal rabbit IgG, 1 mM
Ca2+ and 0.5 mM Mg2+ and rotated for
3 h. The mixture was added to washed beads and rotated at 4 °C over
night. The beads were washed 3x (1 min 4 °C, 3.000 rpm). Proteins were
released from the beads using 27 µl 95 °C, 3x Lämmli-buffer and beads
were removed by centrifuging (5 min 4 °C, 8.000 rpm).