Protein g sepharose

Briefly, cells were collected and lysed with IP buffer containing 0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl, PH 8, 50 mM NaF, and 2 mM EDTA, plus a protease inhibitor mixture (Roche Applied Science). Equivalent amounts of cellular extract were incubated overnight with antibody-coated Protein G Sepharose (GE Healthcare Life, 1 μg antibody and 25 μl Protein G Sepharose for each sample). The immunoprecipitates were washed four times in lysis buffer and eluted by boiling in Laemmli sample buffer (Bio-Rad). The samples were fractionated by SDS-PAGE and transferred to nitrocellulose. Immunoblots were probed with the indicated primary antibodies and visualized using ECL (Thermo).

Anti-rabbit or mouse IgG, or different primary antibodies against endogenous proteins were linked to protein G-sepharose (Amersham, #17-0618-02) by incubating an antibody with sepharose beads in PBS. After being washed, the IgG or specific antibody-conjugated beads were added to cellular extracts and incubated overnight at 4°C. Beads were then washed in TBST buffer (pH7.2, 25 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20). Immuno-complexes were eluted by incubating the beads in 1× loading buffer at 95°C, and subjected to SDS-PAGE.

ChIP using antibodies against trimethyl H3K4me3 (Millipore) was carried out according to a previously described method [49 (link)]. Briefly, cultured cells were cross-linked in 1% formaldehyde for 10 min at 37 °C. After the addition of 1/10 volume of 1.25 M glycine and incubation for 5 min, fixed cells were washed twice with cold PBS buffer. Soluble chromatin was prepared by sonication (Bioruptor sonicator; Cosmo Bio) to an average DNA size of 500 bp in sonication buffer and immunoprecipitated in IP buffer (20 mM Tris–HCl, pH 8.0, 600 mM NaCl, 1 mM EDTA, 0.05% SDS, 1.0% Triton X-100, 20% glycerol, 1.5 μM aprotinin, 10 μM leupeptin, 1 mM DTT and 40 μM MG132). Protein G sepharose (Amersham, USA) blocked with BSA was added and the antibody–chromatin complex recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). Primers for alphoid^tetO repeat (tetO) as well as for a control 5S ribosomal DNA, are listed in Supplementary Table S1. At least three independent ChIP experiments were performed to estimate the level of enrichment.

Total cellular proteins were isolated using RIPA buffer [20 mM Tris–HCl (pH 7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Aliquots of the proteins were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies that were then coupled with the ECL detection system (Amersham Pharmacia Biotech., Tokyo, Japan).
For co-IP, cells were lysed with IP buffer [10 mM Tris–HCl (pH 7.6), 100 mM NaCl, and 10% NP-40] in the presence of 1 mM CaCl₂. Cell lysates were cleared and incubated with anti-S100A4 and NMIIA antibodies, followed by incubation with Protein G-Sepharose (Amersham Pharmacia Biotech). Western blot assay was subsequently performed with anti-S100A4 and anti-NMIIA antibodies.

Cells were lysed in ELB + (150 mM NaCl, 50 mM HEPES (pH 7.5), 5 mM EDTA, 0.3% NP-40, 10 mM β-glycerophosphate, 6% glycerol, 5 mM NaF, 1 mM Na₃VO₄ and Roche protease inhibitor cocktail). Lysates were cleared by centrifugation (13,000 × g, 10 min at 4 °C). Protein levels were equalized by Bradford assay. For immunoprecipitations, 2 μg antibodies were precoupled for 4–12 hours to 20 μl of protein G Sepharose (Amersham Biosciences) and washed with ELB + . Precoupled beads and lysates were incubated for 4 hours at 4 °C and washed three times with 1.0 mL of ice-cold ELB + . All remaining buffer was then removed and beads were resuspended in 50 μL Laemmli sample buffer; 25 μL was separated on SDS-PAGE and blotted on nitrocellulose (0.4 μm pore). Immunoprecipitations of GFP for mass spectrometry were performed with GFP-Trap_A beads (Chromotek), according to the manufacturer’s protocol. Membranes were blocked with 5% ELK in PBS containing 0.1% Tween. Development of blots was performed using the Chemidoc Imaging System (Bio-Rad Laboratories) and quantification was done with the Image Lab (Bio-Rad Laboratories) software.