Protein g mag sepharose xtra beads

Samples were prepared for immunoprecipitation as previously described (Pearring et al., 2015 (link)). Briefly, purified rod outer segments were thawed and their protein content was determined using the DC Protein Assay Kit. Samples were diluted to 0.5 μg/μl protein in the immunoprecipitation buffer (0.1% n-dodecyl-β-maltoside in PBS with protease inhibitor cocktail, Sigma-Aldrich), vortexed, and centrifuged at 108,000 × g for 30 min. The supernatant was removed for use in immunoprecipitation. Protein A Mag Sepharose beads (catalog #28944006, GE Healthcare Life Sciences) and Protein G Mag Sepharose Xtra beads (catalog #28967066, GE Healthcare Life Sciences) were used for immunoprecipitation with rabbit and mouse antibodies, respectively. In both cases, 5 μl beads were incubated with 5 μg of antibody for 2 h under rotation at room temperature. The beads were rinsed and incubated with 12.5 μg of rod outer segment lysate overnight under rotation at 4°C. After the beads were rinsed, bound proteins were eluted by boiling in the SDS-PAGE loading buffer (2% SDS) at 95°C for 10 min and analyzed by Western blotting.

Natural human IgG standards, including IgG1, IgG2, IgG3, and IgG4, were purchased from Abcam (Cambridge, MA, USA). The internal standard used in this study was the SILu™MAB K1 Stable-Isotope-Labeled Universal Monoclonal Antibody, purchased from Sigma-Aldrich (St. Louis, MO, USA). Iodoacetamide (IAA), ammonium bicarbonate, and formic acid were also purchased from Sigma-Aldrich. Dithiothreitol (DTT) was purchased from Merck (Billerica, MA, USA). Protein G Mag Sepharose Xtra beads were obtained from GE (Piscataway, NJ, USA). Chicken serum, used as a blank matrix, was purchased from Thermo Scientific (Sunnyvale, CA, USA). Phosphate-buffered saline (PBS; 10×, sterile solution) was purchased from VWR International (West Chester, PA, USA). Lyophilized trypsin was purchased from Promega (Madison, WI, USA). MS-grade acetonitrile (ACN) was purchased from J.T. Baker (Phillipsburg, NJ, USA).

Five hundred micrograms (500 µg) of total cellular, cytoplasmic, or nuclear proteins were extracted and used for immunoprecipitation by adding 1 µg of anti-Hsp90α antibody in NP-40 cell lysis buffer and incubating at 4 °C for overnight. Ten microliters (10 µL) of Protein G Mag Sepharose Xtra beads (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) were then added and incubated at room temperature for 2 h. After washing steps, precipitated proteins were then eluted by adding 1× sample loading dye and incubated at 95 °C for 10 min. Immunoprecipitated proteins were analyzed by Western blot analysis. For ChIP analysis, cells were fixed with 1% formaldehyde at room temperature for 10 min. After quenching the formaldehyde with 125 mM glycine, the cells were lysed with mammalian cell lysis buffer (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA USA) and chromatins were sonicated into fragments with lengths of 500 base-pairs. The ChIP analysis was then performed according to the previous report [40 (link)]. The c-Myc binding region of BMI1 promoter was detected by SYBR Green based quantitative PCR using the following primer set: forward primer: 5′-CACGGGCCTGACTACACCGACACT-3′; reverse primer: 5′-CACCGCTGAAGGCAGAGTGGAAAC-3′.