Mitochondrial fractions were isolated from WT and OSGEPL1 KO HEK293T cells (1 × 107 cells) using the Mitochondria Isolation Kit (Miltenyi Biotec. K.K.) and subjected to western blotting to measure steady-state levels of protein components in respiratory chain complexes using the Total OXPHOS Rodent WB Antibody Cocktail (an antibody mixture targeting ATP5A, UQCRC2, MTCO1, SDHB, and NDUF88; ab110413, Abcam), an anti-ND5 antibody (ab92624, Abcam), and an anti-ND2 antibody (19704-1-AP, Proteintech). Other antibodies used in this study were as follows: anti-OSGEPL1 (25694-1-AP, Proteintech), anti-GAPDH (6C5, Santa Cruz Biotech), anti-FLAG-tag (1E6, Wako), HRP-conjugated donkey anti-mouse/rabbit IgG (715-035-150/715-035-152, Jackson ImmunoResearch), anti-FLAG M2 affinity gel (A2220, Sigma), and Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11001, ThermoFisher). To monitor YRDC subcellular localization using the anti-FLAG-tag antibody, mitochondrial fractions were carefully washed with 2 mg/mL digitonin (Sigma) to remove the outer membrane with cytoplasmic components.
Ab92624
Manufactured by Abcam
Ab92624 is a lab equipment product offered by Abcam. It is a core component used in scientific research and analysis. The detailed specifications and intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab110413, by Abcam (3 mentions)
Mitochondria isolation kit, by Miltenyi Biotec (2 mentions)
Ab192423, by Abcam (2 mentions)
Ab81215, by Abcam (2 mentions)
Ab14705, by Abcam (2 mentions)
Ab79393, by Abcam (2 mentions)
Dab eqv peroxidase, by Vector Laboratories (2 mentions)
Mirax scan, by Zeiss (2 mentions)
Caseviewer, by 3DHISTECH (2 mentions)
Digitonin, by Merck Group (1 mentions)
Anti flag tag, by Fujifilm (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 6c5, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Ab181848, by Abcam (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Anti gapdh 10494 1 ap, by Proteintech (1 mentions)
Ab21058, by Abcam (1 mentions)
Ab14745, by Abcam (1 mentions)
7 protocols using ab92624
1
Mitochondrial Protein Analysis in HEK293T Cells
2
Mitochondrial Protein Expression Analysis
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Muscle biopsies and cybrid cells were lysed in cell lysis buffer (Beyotime, China). 30‐50 mg protein were run on a 8‐12% sodium dodecyl sulfate‐polyacrylamide gel (SDS–PAGE) at 100 V, 2‐3 h. Proteins were transferred to PVDF (Immobilon‐P PVDF‐Membrane, Millipore, Burlington, MA, USA) for 1‐3 h at 100 V, 4°C. After blocking with 5% dried skimmed milk, the membrane was incubated with various primary antibodies. Primary antibodies were as follows: total OXPHOS antibody cocktail (ab110413, Abcam), anti‐CO4 (CST 4805s, Cell Signaling Technology), anti‐GAPDH (10494‐1‐AP, Proteintech), anti‐ND5 antibody(ab92624, Abcam), anti‐ATP6 antibody (ab192423, Abcam), anti‐ND1 antibody (ab181848, Abcam), Anti‐CYB antibody (ab81215, Abcam), Anti‐VDAC1/Porin antibody (ab158995, Abcam), Anti‐β actin antibody (TA‐09, Zhongshan Jinqiao).
3
Mitochondrial Protein Expression Analysis in B16 Cells
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B16 cells were transfected with Myg1 SiRNA using Dharmafect (SMARTpool:ON-TARGETplus Myg1 siRNA, L-055338-01-0005). After 24 h, the cells were trypsinized and lysed using NP40 lysis buffer supplemented with protease inhibitor cocktail. For detection of mitochondrial protein, 40 μg of cell lysate was resolved on 10% SDS-PAGE gel. Antibody was prepared in 2% skimmed milk solution with following dilutions MT-CO1(Abcam, ab90668, 1:500), MT-ND5(Abcam, ab92624, 1:500), MT-ATP6 (Abcam, ab192423, 1:100), MT-CYB(Abcam, ab81215, 1:100), Myg1 (Abcam, ab122493, 1:2000), Tubulin (Abcam, Ab21058, 1:5000). It was detected using HRP conjugated anti rabbit secondary antibody (1:10 000) and detected through Enhanced Chemiluminescence using ChemiDoc (Syngene).
4
Immunoblotting Analysis of Mitochondrial Proteins
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Total fibroblast cell lysates were subjected to whole protein quantification, separated on 4–12% precast gels (Lonza) by SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and semi-dry transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences). The membranes were blocked in 5% non-fat milk (Bio Rad) in TBS-T (150 mM NaCl, 30 mM Tris base, pH 7.4, 0.1% Tween 20) for 1 h and immunoblotted using primary antibodies (1:1,000 dilution) against CLPP (Abcam, ab56455), MCOLN1 (Abcam, ab28508), MT-ND5 (Abcam, ab92624), NDUFA13 (Abcam, ab110240), NDUFB3 (Abcam, ab55526), NDUFB8 (Abcam, ab110242), TIMMDC1 (Abcam, ab171978) and UQCRC2 (Abcam, ab14745) for 1 h at RT or ON at 4 °C. Signals were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson Immuno Research Laboratories, Code: 111-036-045 and Code: 115-036-062, respectively, 1:5,000 dilution) for 1 h and visualized using ECL (GE Healthcare Life Sciences).
5
Histological Analysis of Dissected Brains
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Dissected brains were fixed in 10% neutral buffered formalin overnight and mounted into paraffin blocks; 5‐μm sections were stained using routine histology techniques. Antibodies used for immunostaining were chicken anti‐GFP (Aves, #1020), anti‐MTND5 (Abcam, #ab92624), anti‐MTCO1 (Abcam, #ab14705), and anti‐MTCO2 (Abcam, #ab79393). Secondary antibodies were conjugated with horseradish peroxidase, and DAB EqV Peroxidase was used as a substrate (all Vector Laboratories). Slides were scanned with MiraxScan (Zeiss), and images were analyzed using CaseViewer (v2.2, 3DHistech) and Fiji/ImageJ (v1.51j, NIH).
6
Histological Analysis of Dissected Brains
7
Mitochondrial Protein Analysis in NSUN2 KO
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Mitochondria were isolated from WT and NSUN2 KO HEK293T cells (1 × 107) using the Mitochondria Isolation Kit (Miltenyi Biotec). Steady-state levels of subunit proteins of mitochondrial respiratory chain complexes were analyzed by immunoblotting with Total OXPHOS Rodent WB Antibody Cocktail (1:250, ab110413, Abcam) and anti-mt-ND5 antibody (1:100, ab92624, Abcam). HRP-conjugated anti-mouse IgG (1:20,000, 715–035-150, Jackson ImmunoResearch) or HRP-conjugated anti-rabbit IgG (1:20,000, 715-035-152, Jackson ImmunoResearch) was used as the secondary antibody.
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