Protein g sepharose bead

C57BL/6 mice were handled in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996, Bethesda, MD, USA). All procedures involving animals were performed in conformity with the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC), College of Medicine, National Taiwan University.
Testes dissected from mice (about 6-weeks-old; weighing about 19 g) were homogenized in ice-cold T-PER tissue extraction reagent (Thermo Scientific, Waltham, MA, USA; 1 testis per 400 µL) containing protease inhibitor cocktail. Lysates were cleared by micro-centrifugation at 13,360× g for 15 min. Solubilized lysates were pre-cleared with protein G sepharose beads (GE Healthcare Biosciences, Piscataway Township, NJ, USA) for 2 h at 4 °C, and then incubated for 16 h at 4 °C with protein G sepharose beads pre-coated with rabbit IgG or rabbit anti-ClC-2 antibody. Beads were gently spun down and washed twice in a wash buffer [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO₃, 20 Tris-HCl, pH 7.5] supplemented with 0.1% Triton X-100, and then twice with the wash buffer. The immune complexes were eluted from the beads by heating at 70 °C for 5 min in the Laemmli sample buffer.

Immunoprecipitation was performed as described previously at 4°C, unless otherwise specified [62 (link)]. Approximately 10⁷ cells in 1 ml of cold 1× RIPA buffer containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) were incubated on ice for 30 min with occasional mixing. Cell lysates were centrifuged at 12,000 × g for 10 min. The supernatant was collected, mixed with primary antibody [calgranulin B (Santa Cruz Biotechnology) or aurora A kinase (Abcam)], and incubated for 2 h with rocking. Prepared protein G Sepharose beads (100 μl; GE Healthcare Life Sciences) were added, incubated on ice for 1 h with rocking and centrifuged at 10,000 × g for 30 s. The supernatant was removed and protein G Sepharose beads were washed five times with 1 ml of cold 1× RIPA to minimize background. Next, 100 μl of 2× SDS sample buffer was added to the bead pellet and heated to 100°C for 10 min. After boiling, immunoprecipitates were centrifuged at 10,000 × g for 5 min, and the supernatant was collected for western blot analysis.

Transfected cells were incubated at 37 °C in the presence of 10 μM MG132 for 24 hrs. Cells were solubilized in ice-cold IP buffer [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO₃, 20 Tris-HCl, pH 7.5, 1 dithiothreitol, 1 PMSF, 1% Triton X-100] containing the protease inhibitor cocktail. Insolubilized materials were removed by centrifugation. Solubilized lysates were pre-cleared with protein G sepharose beads (GE Healthcare Biosciences) for 1 hr at 4 °C, and then incubated for 16 hrs at 4 °C with protein G sepharose beads pre-coated with the anti-Myc or anti-HA antibody. Beads were gently spun down and washed twice in a wash buffer [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO₃, 20 Tris-HCl, pH 7.5] supplemented with 0.1% Triton X-100, and then twice with the wash buffer. The immune complexes were eluted from the beads by heating at 70 °C for 5 min in the Laemmli sample buffer.

K562 cells stably transduced with His-tagged Ex4a(+)WT1 [K562-His-Ex4a(+)] were washed with ice-cold PBS, lysed in RIPA buffer (50mM Tris-HCl pH8.0, 150 mM NaCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA) containing 1 mM PMSF, incubated on ice for 20 min, and then centrifuged at 12.000 x g for 20 min at 4ºC. The supernatants were collected as cell lysates. The cell lysates were precleared with Protein G-sepharose beads (GE Healthcare) for 30 min and then incubated overnight at 4ºC with 3 μg of C-19 or non-immune rabbit IgG coupled to Protein G–sepharose beads. The immunoprecipitates were washed with RIPA buffer and proteins were eluted by boiling in SDS sample buffer. The eluted proteins were separated by SDS-PAGE and analyzed with 6F-H2 antibody (Dako Cytomation, Carpinteria, CA) against the N-terminal region of WT1 protein.

Two hundred to three hundred micrograms of protein lysates, were pre-cleared with 10 μl of the protein G-Sepharose beads (GE Healthcare) for 1 h. The lysates were incubated with antibodies overnight at 4 °C, and then with 25 μl protein G-Sepharose beads for 2 h at 4 °C. The beads were washed with the lysis buffer, and proteins eluted in sample buffer. Flag-tagged RelB was immunoprecipitated with anti-Flag-M2 beads and eluted with Flag peptide (Sigma).