Protein g dynabead

The proteins (200 µg) in the 1% Triton-soluble fraction of the cerebral cortex were mixed with a complex of the R-10G anti-KS antibody and Protein G Dynabeads™ (Thermo Fisher Scientific) in PB containing 0.02% Tween-20 (PB-T) for 30 min at room temperature. The immunocomplexes bound to the Protein G Dynabeads™ were isolated with the DynaMag™ -2 Magnet (Thermo Fisher Scientific). Pretreatment of the samples with chondroitinase ABC was performed in the case of immunoprecipitation followed by the R-10G immunoblot, and molecular identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS).

To carry out immunoprecipitation, 1% Triton X 100-soluble fractions were prepared from tissue homogenates of 100 mg lung lobes of 2- to 3-month-old C57BL/6J mice. The lung lysate was mixed with a complex of the R-10G anti-KS antibody and Protein G Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) in PB containing 0.02% Tween-20 (PB-T) for 30 min at room temperature. The immunocomplexes bound to the Protein G Dynabeads were isolated with the DynaMag-2 Magnet (Thermo Fisher Scientific).

Cells were lysed in Endo-IP buffer with protease inhibitor cocktail (Biotool, B14001) on ice for 30 min, and the lysate was centrifugated at 4 °C for 25 min to remove precipitates. 500 μg total protein was incubated with 5 μg of rabbit IgG or HA antibodies (Sigma, H3663) or Flag antibodies (Sigma, F1804) at 4 °C overnight with rotation. Lysate and antibody mix was incubated with 30 μl protein G Dynabeads (Thermo Fisher Scientific, 10003D) for 2 h at room temperature with rotation. Beads were washed with lysis buffer and eluted. The eluent was analyzed by western blotting. To detect protein ubiquitination, 500 μg total protein was incubated with 5 μg of rabbit IgG or ZSCAN4 antibodies (Millipore, AB3430) at 4 °C overnight with rotation. Lysate and antibody mix were incubated with 30 μl protein G Dynabeads (Thermo Fisher Scientific, 10003D) for 2 h at room temperature with rotation. The eluent was later blotted with ubiquitin antibody (CST, #3936).

1x10⁷ cells were collected after transfection for in vitro co-immunoprecipitation and 2 pairs of P21 or P28 juvenile testes were used for in vivo co-immunoprecipitation. Cells or testes were lysed in 1 ml RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1% Trion X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with 1mM PMSF. For immunoprecipitation (IP), 1.5% of the lysates were used as inputs. The remaining lysates were pre-cleared with 15 μl protein G Dynabeads (Thermo Fisher Scientific) for two hours, incubated with 1–2 μg primary antibodies at 4°C for 1 hour, and then incubated with 30 μl protein G Dynabeads overnight. The immunoprecipitated complexes were washed with the RIPA buffer four times and boiled in 30 μl 2× SDS-PAGE loading buffer for 10 min. 20 μl of supernatants were resolved by SDS-PAGE, transferred onto nitrocellulose membranes using iBlot (Invitrogen), and immunoblotted with indicated antibodies. The primary and secondary antibodies used for co-IP and western blot analyses were listed in S1 Table. Band quantification was performed with ImageJ (Version 1.51).

ChIP was performed as previously reported (Brown et al., 2011 (link)). Briefly, chromatin was crosslinked with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature and quenched with 0.125 M glycine (Sigma-Aldrich). Cells and nuclei were subsequently lysed and chromatin was sonicated to fragment DNA to about 200–500 bp on a Bioruptor Pico sonication device (Diagenode). Sonicated chromatin was pre-cleared with same-host IgG and protein G Dynabeads (Thermo Fisher), 100 µg of cleared chromatin (protein) was incubated with 2 µg of the following antibodies overnight at 4°C: H3K27ac (Abcam, ab4729), H3K4me1 (Abcam, ab8895), H3K27me3 (Active Motif, 39155), and H3K4me3 (Merk, 05-745R), after which complexes captured with 30 µl of protein G Dynabeads (Thermo Fisher). Complexes were washed, RNAse A (Thermo Fisher) and Proteinase K (Sigma-Aldrich) treated, and DNA was purified by phenol-chloroform extraction and precipitated with GlycoBlue (Thermo Fisher), sodium acetate (Thermo Fisher), and ethanol (Sigma-Aldrich). A sonicated chromatin sample (1%) was also collected as input for normalisation and 10 ng of DNA were used for ChIP-seq library preparations.