2.4 Immunoprecipitation
Cultured HDF cells were collected and subjected to protein extraction.
Briefly, RIPA buffer was added to the cells and incubated at 4°C for 10
minutes. Cells were detached by vigorous pipetting and followed by
continuous rapid syringe outing. The lysate was collected through
centrifugation at 12,000 rpm for 5 minutes at 4°C. To the 1 ml of
protein lysate, 2µg of primary antibody (anti-trf2 antibody) was added
and incubated for 2 hours at 4°C. In the mixture, 20µl of agarose beads
were added to the lysate and incubated at 4°C on rocker for 2 hours.
After incubation, protein lysate was centrifuged at 2,500 rpm for 5 min
at 4°C. The pellet was washed three times with RIPA buffer and
re-suspended in 40µl of 2X protein sample buffer and incubated at 100 °C
for 5 minutes. The immunoprecipitated protein was collected and
estimated by Lowry’s method and stored accordingly at -20°C for
immunoblotting experiments.