Cells (5 × 105) were washed with PBS, resuspended in 400 μl of 4 mg/ml digitonin (Sigma) and protease inhibitor cocktail (PIC; Sigma) and incubated on ice for 10 min. Samples were washed twice with 1 ml cold PBS and centrifuged at 10,000g for 10 min at 4 °C. Pellets were resuspended in 1× NativePAGE Sample buffer (Invitrogen) containing 1% DDM (Invitrogen #BN2005) and PIC. After 5 min on ice, the lysates were spun at 22,000g for 30 min at 4 °C. G250 (10 μl; Invitrogen) was added to the supernatant and 25 μl of solubilized complexes were loaded onto a 3–12% native precast gel at 4 °C (Invitrogen). Gels were run in dark blue cathode running buffer (Invitrogen) for 30 min and subsequently in bright blue cathode running buffer (Invitrogen) for 75 min. For OXPHOS complex detection, the total OXPHOS Blue Native WB Antibody Cocktail (abcam, catalogue no. ab110412; dilution 1:2,000) and anti-NDUFS3 antibody (Genetex, catalogue no. GTX105835; dilution 1:1,000) were used. Uncropped and unprocessed scans are provided in Supplementary Fig. 1 .
Gtx105835
Manufactured by GeneTex
Sourced in United States
GTX105835 is a laboratory equipment product from GeneTex. It is a precision scientific instrument designed for specific laboratory applications. The core function of this product is to perform accurate measurements and data collection. No further details on the intended use or specific applications of this product are available.
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Lab products found in correlation
2 protocols using gtx105835
1
Native PAGE Analysis of OXPHOS Complexes
2
Immunofluorescence Assay for RMC Cells
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RMC cells at a density 4 × 104/mL prepared with 2% charcoal FBS medium were seeded onto 1 µ-Slide 4 Well Chamber Slide (ibidi 80284, poly-L-lysine coated) and incubated for 24 h at 37 °C under 5% CO2 atmosphere until adhered. The medium was removed and replaced with Bic (0, 60 μM)-containing medium prepared from 2% charcoal FBS medium and incubated for additional 24 h. The medium was removed, the cells were rinsed twice with PBS, and the washings were discarded. A paraformaldehyde solution (4%) was added to fix the cells at ambient temperature for 20 min. After antigen retrieval with citrate buffer (pH 6.0) for 15 min, 10% BSA blocking solution was applied and left at ambient temperature for 1 h. Primary antibody Complex 1 (GTX105835, GeneTex, Inc., Irvine, CA, USA) in BSA-PBST was added and kept at 4 °C overnight. The cells were rinsed thrice with PBS, and anti-rabbit secondary antibody in 1% BSA-PBST was added and left to stand at room temperature for one hour. The cells were rinsed thrice with 1% PBS-PBST. The washings were removed, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) in PBST was added, left at ambient temperature for 15 min. The cells were rinsed thrice with PBS and then subjected to fluorescence imaging.
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