Protein g sepharose

Transfected HEK293 cells were fixed for 4 min with 0.5% formaldehyde and lysed in RIPA buffer with 1% Triton X-100. Lysates were sonicated to obtain chromatin fragments of less than 1 kb. Lysate supernatants were incubated overnight with 2 μg of antibody immobilized on protein G-Sepharose (Qiagen) over night at 4°C. Pellets were washed and chromatin was eluted from the beads and crosslink was reversed. Hybridization with the biotin PCR-labeled TTAGGG probe or an Alu probe was performed and the biotinylated probe was detected with ABC kit (ThermoFisher Scientific). The intensity of the signal from telomere repeats was normalized to the signals of input DNA and Alu repeats. The antibodies used were TRF1 (Santa Cruz), TRF2 (Santa Cruz), TNKS (ThermoFisher Scientific), and GFP (Sigma).

HEK293 cells were lysed in isotonic buffer with 1% Triton X-100. Lysate containing 400 μg of protein was incubated with 2 μg of antibody immobilized on protein G-Sepharose (Qiagen) over night at 4°C. The beads were washed with lysis buffer and proteins were eluted by heating at 95°C for 5 min in SDS loading buffer supplemented with 100 mM DTT.
Samples were subjected to SDS-PAGE and electro-blotted onto Immobilon-P (Millipore). Specific proteins were immuno-detected and visualized using enhanced chemiluminescence reagent (Amersham Biosciences) and X-ray film or GeneGnome chemiluminescence detection system (Syngene). The signal intensity was quantified using AlpfaEaseFC software (Alpha Innotech). For normalization of signals, Input or control signals from the same membrane were used. The antibodies used were BARD1 N19 (Santa Cruz), BARD1 H300 (Santa Cruz), TRF1 (Santa Cruz), TRF2 (Santa Cruz), TNKS (ThermoFisher Scientific), ABC biotin detection kit (ThermoFisher Scientific).