To examine countin protein expression levels during aggregation, a Western blot was performed with anti-countin antibody. Cells were resuspended in SDS Laemmli buffer, and boiled for 3 min. Subsequently, the samples were run in a 12% SDS-polyacrylamide gel and Western blots were developed using an ECL Western blotting kit (Bio-Rad). Rabbit anti-countin antibodies were used at 1: 3000 dilution.
Ecl western blotting kit
Manufactured by Bio-Rad
Sourced in United States
The ECL Western blotting kit is a laboratory equipment designed for performing Western blot analysis. It provides the necessary reagents and materials to detect and quantify specific proteins in a sample using chemiluminescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Mitotracker cmxros, by Thermo Fisher Scientific (1 mentions)
Gapdh mab374, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay reagent kit, by Beyotime (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab37168, by Abcam (1 mentions)
Ab37150, by Abcam (1 mentions)
Ab175781, by Abcam (1 mentions)
Ab175775, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti gapdh monoclonal antibody, by Biosynth (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
9 protocols using ecl western blotting kit
1
Quantifying Countin Protein Levels
2
Western Blot Analysis of Proteins
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Cells were lysed in lysis buffer containing 150 mM NaCl, 1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris (pH 8.0) and a protease inhibitor cocktail (Roche Molecular Biochemicals), as described previously.20 (link) The proteins from FaDu cells were electrotransferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA, USA). Detection of specific proteins was carried out with an ECL Western blotting kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.
3
Mitochondrial Dynamics Protein Analysis
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Antibodies: DRP1(H00010059-M01) and MFN2 (H00009927-M01) from Abnova; OPA1 (612607) and TIM23 (611222) from BD Biosciences; HSP60 (SC-13966) from Santa Cruz; ATG5 (12994), ATG7 (8558), DRP1-phosphoS616 (3455), and TOM20 (42406) from Cell signaling; NDUFA9 (ab14713), SDHA (ab14715), UQCRC2 (ab14745), COX IV (ab14744), ATP5A (ab1801) and CS (ab129095) from Abcam; GAPDH (MAB374) from Millipore; LC3 (L7543), ACTIN (SAB00001) and TUBULIN (T6557) from Sigma-Aldrich; Mitotracker CMX Ros (M7512) from ThermoFisher Scientific. Horseradish peroxidase-conjugated secondary antibodies from Jackson Immuno Research (Mouse 115-035-146, Rabbit 111-035-144). ECL western blotting kit from Biorad or Thermo Scientific Pierce. Fluorescent secondary antibodies anti-rabbit (926-32210) or anti-mouse (926-68071) from Licor.
4
Western Blot Analysis of Protein Expression
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Total proteins from transfected A549 cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China). The BCA Protein Assay Reagent Kit (Beyotime) was used to measure protein concentrations. Proteins were loaded onto 10% SDS-PAGE, then transferred onto PVDF membranes (Bio-Rad) and blocked with 5% skimmed milk for 2 h at room temperature. Subsequently, the membranes were incubated with primary antibodies (CDK2, 5 μg/ml, ab6433; cyclinE1, 1: 2000, ab71535; P21, 1: 3000, ab227443; MMP2, 1 μg/ml, ab37150; MMP9, 1: 1000, ab38898; Bcl-2, 1: 500, ab196495; Bax, 1: 500, ab53154; cleaved caspase3, 1: 500, ab49822; IL-6, 1: 500, ab6672; JAK2, 1: 1000, ab39636; p-STAT3, 1: 1000, ab30647; HIF-1α, 1: 1000, ab216842; STAT3, 1: 5000, ab119352; Beclin-1, 1 μg/ml, ab62557; p62, 1: 2000, ab155686; ATG14, 1 μg/ml, ab139727 and GAPDH, 1 μg/ml, ab37168, all from Abcam, overnight at 4°C. After washing 3 times with TBST, the membranes were incubated with goat anti-rabbit IgG (1: 10 000, ab175781, Abcam) or goat anti-mouse IgG (1: 10 000, ab175775, Abcam) secondary antibodies at room temperature for 2 h. ECL Western blotting kit (Bio-Rad lab, USA) was used to visualize the protein bands. GAPDH was used for an internal control.
5
Western Blot Analysis of Cell Lysates
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Total cell or cytoplasmic lysates were prepared as described previously (30 (link)). Cell lysates (10 μg) were resolved on a 7, 10 or 4–15% gradient SDS-polyacrylamide gel and analysed using an ECL Western blotting kit (Bio-Rad). The PVDF blots were probed with appropriate primary antibody at the dilutions indicated below and detected with peroxidase-conjugated secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (Pierce). Primary antibody dilutions were: anti-hMALT1 (Cell Signaling Technology), 1:3000; anti-hHMGA1 (Abcam), 1:10 000; anti-β-actin HRP-conjugated monoclonal antibody (Santa Cruz Biotechnology), 1:20 000 and anti-GAPDH monoclonal antibody (Fitzgerald), 1:20 000. Secondary antibody dilution was: goat anti-rabbit IgG HRP (ThermoScientific), 1:3000. The fluorescence signals and intensity of detected bands were captured and quantified by an imaging system (Syngene) and the software GeneTools (Syngene), respectively. Enzyme-linked immunosorbent assays (ELISA) for IL-6 in culture medium were performed according to the manufacturer's instructions (Affymetrix, eBioscience).
6
Western Blot Analysis of EDA-Fibronectin
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Cells were lysed in Radio-Immunoprecipitation Assay (RIPA) Buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate SDS) containing 1:100 phenylmethylsulfonyl fluoride and phosphatase inhibitors (Sigma). Prepared samples were heated at 100°C for 4 min; for each sample the same amount of total protein (10 μg) was added to a well of 4–15% mini-protean TGX precast gels polyacrylamide gel (Bio-Rad Hercules, CA, USA) and resolved by SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane (Bio-Rad). The membranes were blocked for 1 h in Tris-buffered saline (10 mM Tris-HCl pH7.5 and 0.15 M NaCl) containing 0.1% (v/v) Tween 20 and 5% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich), and then probed at room temperature (RT) for 1 h with primary antibodies against human EDA-fibronectin (diluted 1:400, Abcam). Immunoreactive bands were detected with IgG horseradish peroxidase-conjugated secondary antibodies (anti-mouse diluted 1:1,000) (Dako, Glostrup, Denmark) and visualized by enhance chemiluminescence detection reagents ECL Western blotting kit (Bio-Rad) according to the manufacturer´s instructions in a luminescent image analyzer (Chemi Doc Bio-Rad) and were then scanned for densitometry analysis (Image Lab Bio-Rad).
7
Western Blot Analysis of DNA Damage
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The Western blot analysis was performed as previously described. U87-EGFRvIII cells were cultured in DMEM supplement with 10% FBS at 37°C in a 5% CO2/95% air atmosphere. The cells were planted in a 6-well plate and treated with DMEM, AuNP or U2-AuNP, respectively. After 24h, cells were lysed in RIPA buffer supplemented with protease inhibitors. After protein quantification with a BCA protein assay kit (Thermo Scientific), 20µg protein was detected by 8% SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were incubated with the following specific antibodies: anti-pEGFR (tyr1038), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pH2AX (S139), anti-pChk2 (T68), anti-Chk2 (Cell Signaling Technology, Danvers, MA), anti-pATM (Ser1981), anti-ATM (Cell Signaling Technology, Danvers, MA), anti-53BP1 (Cell Signaling Technology, Danvers, MA) and anti-β-actin (Boster Biological Technology, China). A secondary HRP-conjugated antibody was used for immunodetection, and an ECL Western blotting kit (Bio-Rad) was used to visualize the protein bands. Finally, the results were analyzed with ImageJ software.
8
Mitochondrial Protein Profiling
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Antibodies: DRP1, MFN1, MFN2 (Abnova); NDUFA9, UQCRC2, α subunits CV, actin (Abcam); COX IV, SDHA (ThermoFisher Scientific); OPA1 (BD Biosciences); tubulin (Sigma-Aldrich); VDAC (Biovision); horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch or Alexafluor). ECL western blotting kit from Biorad or Thermo Scientific Pierce. The other reagents from Sigma-Aldrich.
9
Western Blot Protein Quantification
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The preparation of total cell lysates and the analysis of western blots were carried out as described previously [30] . Electrophoresis of an equivalent amount of cell lysate (30 µg) on 6~15% percent SDS-PAGE gels was performed, followed by electrotransfer to a PVDF membrane. As directed by the manufacturer, individual proteins were detected with an ECL Western Blotting Kit (Bio-Rad). ImageJ software was used for densitometry, and the density of the protein of interest was normalized to that of βactin using arbitrary densitometric units.
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