Rfp trap agarose beads

RFP-fused proteins were immobilized on RFP-Trap® agarose beads (ChromoTek) and used for co-precipitation assays according to the manufacturer’s protocol. Briefly, cell lysates from co-transfected HEK293T cells (RFP-tagged proteins or RFP alone together with HA- or SF-tagged proteins, respectively) were suspended in lysis buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40), spun and the supernatant was diluted to 1 mL in dilution buffer (10 mM Tris–Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). Fifty microliters were separated as input (total cell lysate) and samples were added to equilibrated beads for 2 h at 4°C under constant shaking. After washing, precipitated protein complexes were eluted with SDS-sample buffer and subjected to SDS–PAGE and western blotting.

Whole cell lysates were prepared by lysing cells with a buffer containing 20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 10% glycerol, and 1 × protease inhibitor cocktail (Roche). Whole cell lysates were incubated with GFP-Trap-A beads or RFP-Trap Agarose beads (ChromoTek) to enrich and isolate Y/RFP or Y/RFP-tagged proteins as described previously (Guo et al., 2017 (link)). Whole cell lysates were incubated with SUMO-2/3 affinity agarose beads (ASM24; Cytoskeleton) at 4°C for overnight to enrich and isolate SUMO-2/3 or SUMO-2/3 conjugates. Whole cell lysates were incubated with glutathione-sepharose 4B beads (Generon) to enrich and isolate GST or GST-tagged proteins as described previously (Guo et al., 2017 (link)). To immunoprecipate endogenous Mff, Bcl-x_L or Drp1, whole cell lysates were incubated overnight with an Mff rabbit polyclonal antibody (17090-1-AP, Proteintech), a Bcl-x_L rabbit monoclonal antibody (54H6, Cell Signaling) or a Drp1 rabbit monoclonal antibody (D6C7, Cell Signaling), pre-conjugated to protein-A beads (Sigma), respectively. The protein-A beads were spun down, washed three times and resuspended in SDS sample buffer for immunoblot analysis.

Immunoprecipitation of SIRT2-GFP and mCherry-SIRT2 was performed using GFP-Trap® and RFP-Trap® agarose beads (Chromotek) respectively, according to the manufacturer’s protocol. Briefly, 2 × 10⁶ HeLa cells were seeded in a 10 cm dish and transfected with tagged-SIRT2 or empty pEGFP-N1/pmCherry-C1. 24 hours post transfection cells were collected using PBS with 5 mM EDTA, washed once in PBS, and lysed in 200 uL RIPA buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.1% SDS; 1% deoxycholate; 1% Triton X-100) supplemented with 25U Benzonase nuclease (Millipore), 1x Complete EDTA-free protease inhibitor cocktail (Roche) and 1x PhosSTOP phosphatase inhibitor (Roche) and 1 mM PMSF. Lysates were then diluted with 600 uL of wash/dilution buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA). 40 uL was removed for input and the remaining lysate was incubated with GFP-Trap® agarose beads at 4 °C with agitation for 1 hour. The beads were washed twice in wash buffer and once in wash buffer containing 300 mM NaCl. Proteins were eluted by boiling beads in 50 uL 2x Laemmli buffer with 100 mM DTT.