15 μg of Mitochondrial lysates and supernatant fraction (cytosolic) were resolved by 7% SDS-PAGE for higher molecular proteins and 12% SDS-PAGE for small molecular proteins, then transferred to polyvinylidene difluoride membrane (PVDF). Membranes were blocked with 5% nonfat dried milk in TBST, incubated overnight at 4 °C with the indicated primary antibodies (HA epitope, BioLegend, 1:1000 dilution), SLC4A11 [37 (link)] (Custom, 1:500), TOM20 (SCBT, 1:1000), TIM23 (BD, 1:1000), VDAC (CST, 1:1000), UCP2 (CST, 1:1000), GLS1 (CST, 1:1000), Cytochrome c (Abcam, 1:1000), and α-tubulin (SCBT, 1:1500), washed three times in TBST (138 mM NaCl, 20 mM Tris, and 0.5% Tween 20, pH 7.6) for 10 min each, and incubated with secondary antibodies for 1 h at room temperature. After washing the membrane with TBST three times for 10 min each, the immunoreactive bands were visualized by enhanced chemiluminescence, ECL buffer (#34578, Thermo Fisher Scientific) using Gel Doc XR + system (Bio-Rad, Hercules, CA).
α tubulin
Manufactured by SCBT
Sourced in United States
α-tubulin is a protein that is a crucial component of the cytoskeleton in eukaryotic cells. It is a member of the tubulin family and forms the building blocks of microtubules, which are essential for cell division, intracellular transport, and cell shape maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc xr system, by Bio-Rad (2 mentions)
Ecl buffer, by Thermo Fisher Scientific (2 mentions)
Cytochrome c, by Abcam (1 mentions)
Tim23, by BD (1 mentions)
Tom20, by SCBT (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Ha tag, by BioLegend (1 mentions)
Hsp90, by SCBT (1 mentions)
Hsc70, by SCBT (1 mentions)
Tom70, by SCBT (1 mentions)
Gapdh, by SCBT (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
γ h2ax, by Abcam (1 mentions)
Cyclin a, by Abcam (1 mentions)
β actin, by SCBT (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Ps473 akt, by Cell Signaling Technology (1 mentions)
Alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
4 protocols using α tubulin
1
Mitochondrial Protein Expression Analysis
2
Mitochondrial Protein Expression Analysis
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Whole cells/isolated mitochondria from cells were lysed in ice-cold 1× RIPA buffer (#9806; Cell Signaling Technology) with addition of 1 mM PMSF (#8553; Cell Signaling Technology) immediately before use. Then, 15 µg of lysates for whole cells, mitochondrial or cytosolic fraction (supernatant) was resolved by SDS-PAGEs and then transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% nonfat dried milk in TBST and incubated overnight at 4°C with the indicated primary antibodies. Secondary antibodies were applied for 1 hour at room temperature. Primary antibodies used were HA tag (#901501; BioLegend), SLC4A11 (custom33 (link); detects all isoforms SLC4A11-A, SLC4A11-B, and SLC4A11-C; see Supplementary Fig. S4 b), HSP90 (#sc-13119; SCBT, Dallas, TX, USA), HSC70 (#sc-7298; SCBT), VDAC (#ab15895; Abcam, Cambridge, MA, USA), ANT (#ab102032; Abcam), RISP (#sc-271609; SCBT), COXIV (#4850; CST, Danvers, MA, USA), TOM70 (#sc-390545; SCBT), α-tubulin (#sc-8035; SCBT), GAPDH (#sc-32233; SCBT), and β-actin (#A5441; Sigma-Aldrich). The immunoreactive bands were visualized by enhanced chemiluminescence, ECL buffer (#34578; Thermo Fisher Scientific), using the Gel Doc XR+ system (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of DNA Repair Proteins
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Samples for western blot assays were prepared as described previously3 (link). The following primary antibodies were used: Rad51 (diluted 1:2,000; Cat. No. sc-8349; SCBT); Rad54 (diluted 1:2,000; Cat. No. sc-374598; SCBT); PCNA (diluted 1:2,000; Cat. No. sc-56; SCBT); Plk1 (diluted 1:3,000; Cat. No. sc-17783; SCBT); Oct3/4 (diluted 1:3,000; Cat. No. sc-5279; SCBT); Chk2 (diluted 1:3,000; Cat. No. sc-9064; SCBT); α-Tubulin (diluted 1:5,000; Cat. No. sc-8035; SCBT); β−Actin (diluted 1:10,000; Cat. No. ab8226; Abcam); Cyclin A (diluted 1:2,000; Cat. No. sc751; SCBT); CENP-F (diluted 1:1000; Cat. No. ab5; Abcam); γH2AX (diluted 1:2,000; Cat. No. 2577; CST); TopBP1 (diluted 1:3,000; Cat. No. sc-271043; SCBT); and Exo1 (diluted 1:1,000; Cat. No. MS-1534; Thermo Fisher). Immunoblot signals were developed with a WEST-ZOL detection system (Cat. No. 16024; iNtRON Biotechnology). The relative amount of each protein was quantified using ImageJ software.
4
Comprehensive Protein Analysis in Cell Lines
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TFP, dimethylsulfoxide (DMSO), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3′,5,5′-Tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO). Propidium iodide (PI) was obtained from Fisher Scientific. BDP and antibodies specific to FOXO3 (FKHRL1) N-16 (1:500 dilution) and H-144 (1:1000 dilution), p53-pS15 (1:1000 dilution), c-Myc (1:1000 dilution), α-Tubulin (1:2000 dilution), PARP1 (1:1000 dilution), SOD2 (1:2000 dilution), and Lamin A/C (1:1000 dilution) were purchased from SCBT. Antibody against phospho-H2AX Serine-139 (1:1000 dilution) was obtained from Millipore (Billerica, MA). Specific antibodies against phospho-FOXO3-S318/321 (#9465, 1:250 dilution), Akt-pS473 (1:1000 dilution), Akt (total, 1:1000 dilution), and p27Kip1 (1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against FOXO3 (2071-1 and 3280-1) (1:1000 dilution) were obtained from Epitomics (Burlingame, CA). Antibody against β-Actin (1:3000 dilution) was purchased from Sigma. Antibody against GAPDH (1:1000 dilution), Alexa 488- and Alexa 594-conjugated secondary antibodies were obtained from Thermo-Fisher Scientific (Waltham, MA). Goat anti-mouse IgG and goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA).
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