Ovarian proteins were extracted using protein lysis buffer (Sangon Biotech, Shanghai, China) and quantified using a protein quantitation kit (Bio‐Rad). Next, equal amounts of protein were loaded per sample and separated by 10% SDS‐PAGE electrophoresis before being transferred to a PVDF membrane. The membrane was blocked with 5% milk in TBST with Tween‐20 for 1 h, and then incubated with primary antibodies at 4°C overnight. Specifically, the total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam). After several washes, the membrane was incubated with corresponding secondary antibodies for 1 h at room temperature. Finally, protein bands were visualized using an enhanced chemiluminescence detection system (Bio‐Rad), and protein expression levels were calculated by normalizing to Gapdh levels using Image J software.
Oxphos rodent wb antibody cocktail
Manufactured by Abcam
Sourced in United Kingdom
The OXPHOS Rodent WB Antibody Cocktail is a set of antibodies designed for the detection of the key subunits of the oxidative phosphorylation (OXPHOS) complexes in rodent samples using western blotting. The cocktail contains antibodies specific to the NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome c reductase (Complex III), cytochrome c oxidase (Complex IV), and ATP synthase (Complex V) complexes.
Automatically generated - may contain errors
Lab products found in correlation
Bnip3 3769, by Cell Signaling Technology (2 mentions)
Lck 2752, by Cell Signaling Technology (2 mentions)
P tyr 100 9411, by Cell Signaling Technology (2 mentions)
Pgc1α h 300, by Santa Cruz Biotechnology (2 mentions)
Actin c4, by Santa Cruz Biotechnology (2 mentions)
Protein lysis buffer, by Sangon (1 mentions)
Protein quantitation kit, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
Ab15895, by Abcam (1 mentions)
Ab110413, by Abcam (1 mentions)
Complete mini tablet, by Roche (1 mentions)
Ab8245, by Abcam (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Tubulin and flag, by Merck Group (1 mentions)
P616 drp1, by Cell Signaling Technology (1 mentions)
Senp2, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Flag hrp, by Merck Group (1 mentions)
Gapdh, by Fortis Life Sciences (1 mentions)
Histone h4, by Merck Group (1 mentions)
8 protocols using oxphos rodent wb antibody cocktail
1
Ovarian Protein Expression Profiling
2
Immunoblotting Protein Detection Protocol
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Immunoblotting was performed as previously described54 (link). Actin (C4) and PGC1α (H-300) antibodies for immunoblots were obtained from Santa Cruz Biotechnology, p-AKT (S473) (D9E), pS6 (S235/236) (D57.2.2E), BNIP3 #3769, LCK #2752, p-Tyr-100 #9411 from Cell Signaling, and CV- ATP5A, CIII- UQCRC2, CII- SDHB from Total Rodent OXPHOS Rodent WB antibody cocktail from Abcam. Immunoblots were detected via standard secondary detection and chemiluminescent exposure to film. Digitally captured films were analyzed densitometrically by ImageJ software.
3
Isolation and BN-PAGE analysis of mitochondrial complexes
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Mitochondria were isolated as previously described (61 (link)). Mitochondria were solubilized in 1% digitonin and separated on 4–10% acrylamide–bisacrylamide BN-PAGE gels as previously described (62 (link)) and detected by Western blot using a total OXPHOS Rodent WB Antibody Cocktail (ab110413; Abcam).
4
Immunoblot Analysis of Mitochondrial Proteins
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Immunoblots were performed on frozen LV that was homogenized in tissue lysis buffer (HEPES, pH 7.4, 20 mmol/L, B‐glycerol phosphate 50 mmol/L, EGTA 2 mmol/L, DTT 1 mmol/L, NaF 10 mmol/L, NaVO4 1 mmol/L, Triton X‐100 1%, glycerol 10%, and 1 protease inhibitor complete mini tablet, EDTA free, 20 mL [Roche]). Total protein (25 μg) was separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Blots were incubated with mouse total OXPHOS Rodent WB Antibody cocktail (containing 5 antibodies, 1 each against complex I subunit [NDUFB8], complex II subunit B [SDHB], complex III core protein 2 [UQCRC2], complex IV subunit I [MTCO1], and complex V alpha subunit [ATP5A]) (Abcam; ab110413), anti‐VDAC1 (Abcam; ab15895), and anti‐GAPDH (Abcam; ab8245) and detected with the use of the Licor Odyssey 2‐color infrared imaging system.
5
Quantification of Protein Expression in Islets
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Proteins were extracted from isolated islets or NIT-1 cells using RIPA lysis buffer. Proteins (15–20 μg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, Clifton, NJ, USA). After incubation with specific antibodies, the membrane bands were visualized by Amersham Imager 680 Blot and Gel Imagers (GE Healthcare Life Sciences, Marlborough, MA, USA). Antibodies against SENP2 (Santa Cruz Biotechnology Inc.), p616 DRP1 and DRP1 (Cell Signaling Technology, Danvers, MA, USA), GFP/YFP (Arigo Biolaboratories, Hsinchu City, Taiwan), GAPDH (Merck Millipore, Burlington, MA, USA), tubulin and Flag (Sigma-Aldrich) and OXPHOS Rodent WB antibody cocktail (Abcam, Cambridge, UK) were used for western blotting.
6
Comprehensive Antibody Panel for Drosophila
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We utilised the following primary antibodies directed against Drosophila Mle (in-house), glorund (DSHB 5B7_C), Squid (DSHB 2G9-c), Rump (DSHB 5G4), Histone H3 (Active Motif 39763) and Beta Actin (Santa Cruz (I-19) sc-1616) as well as antibodies recognising human DHX9 (Abcam ab183731), HNRNPM1–4 (Santa Cruz sc-20002), EIF-4A1 (Abcam EPR14506/Ab185946), KHDRBS1 (Sigma S9575), Beta Actin (Santa Cruz (I-19) sc-1616), Histone H3 (Active Motif 39763), histone H4 (Millipore 05–858), OXPHOS Rodent WB Antibody Cocktail (Abcam ab110413- to detect Complex I member NDUF88, Complex II member SDHB, Complex III member UQCRC2, Complex IV member MTCO1 and Complex V member ATP5A1), OXPHOS complex II member SDHA (Invitrogen 459200), TOMM20 (Santa Cruz sc-11415), XRCC5 (Invitrogen MA5–15873), MSH6 (Cell Signalling 5424P), GAPDH (Bethyl A300-641A), HUR (3A2) (Santa Cruz 5261) and FLAG HRP (Sigma A8592). All antibodies were used at a dilution of 1:1000 in 5% fat free milk powder dissolved in 0.3% Tween-20 phosphate buffered saline (Supplementary Figs 19 –21 ).
7
Multiplex Western Blot Analysis of Metabolic Regulators
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For detection of Gclc (Santa Cruz Biotechnology sc-390811), Pdha-1 (Abcam ab168379), DRP1 (Abcam ab184247), eIF4E (Abcam ab33766), p-eIF4E (Abcam ab76256), 4E-BP1 (Cell signaling 9452), p-4E-BP1 (Cell signaling 2855), DRP1 (Abcam ab184247), mTOR (Cell Signaling 2972), p-mTOR (Ser2448) (Cell Signaling 2971), p70 S6 (Cell Signaling 9202), p-p70 S6 (Thr421/Ser424) (Cell Signaling 9204), 1–5 × 106 cells were lysed with lysis buffer (CST 9803S) and protein/phosphatases inhibitors as per manufacturer’s protocol and blotted as described44 . Briefly, cells were washed and lysed on ice for 30 min and spun. Supernatants were assayed for total protein concentration with Bradford assay (BioRad 5000006) and 50–100 µg of protein was mixed with loading buffer and loaded in a 12 or 16% gradient gel (Thermo 1Fisher XP00162). Proteins were transferred onto a NC or PVDF membrane (Fisher Scientific) and blocked with 5% milk for 1 h. OXPHOS complexes were revealed with total OXPHOS Rodent WB Antibody Cocktail (Abcam ab110413). Lysis was carried out with the addition of lauryl maltoside at 1.5%.
8
Immunoblotting Protein Detection Protocol
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