Total proteins were extracted from the cells using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) with enzyme inhibitor cocktail (Complete; Roche, Basel, Switzerland). After being lysed on ice for 30 min, the lysate was centrifuged at 13,000 × g for 20 min, and the supernatant was collected for experiments. For the western blot analysis, 25 µg of extracts were separated using 10% SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA) and incubated with anti-SIRT1 (mouse monoclonal antibody; 1:1,000; Cat. no. 8469), acetylated-p53 (Lys382; rabbit polyclonal antibody; 1:1,000; Cat. no. 2525), p53 (rabbit monoclonal antibody; 1:1,000, Cat. no. 2527), Bax (rabbit monoclonal antibody; 1:1,000; Cat. no. 5023), Bcl-2 (rabbit monoclonal antibody; 1:1,000; Cat. no. 4223) (Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-GAPDH (mouse monoclonal antibody; 1:500; Cat. no. sc-365062; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies for 24 h at 4°C. GAPDH was used as internal control. The blots were developed using a chemiluminescent substrate kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Intensity of the bands was detected and analyzed using Quantity One analyzing system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Quantity one analyzing system
Manufactured by Bio-Rad
Sourced in United States
The Quantity One analyzing system is a lab equipment product designed for image analysis and quantification. It provides core functionalities for processing and analyzing digital images generated from various life science applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Cytiva (2 mentions)
Mouse anti β actin monoclonal antibody, by Abcam (2 mentions)
X ray film, by Kodak (2 mentions)
Novex bis tris sds acrylamide gels, by Thermo Fisher Scientific (2 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti sirt1, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Qproteome mitochondria isolation kit, by Qiagen (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
Rabbit anti mecp2 polyclonal antibody, by GeneTex (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Halt protease inhibitor single use cocktail 100x, by Thermo Fisher Scientific (1 mentions)
6 protocols using quantity one analyzing system
1
Western Blot Analysis of Protein Expression
2
Mitochondrial Protein Expression Analysis
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Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer. Extracts of mitochondrial fractions were prepared using the Qproteome Mitochondria Isolation Kit (Qiagen) according to the manufacturer’s instructions. Protein concentration was detected by BCA assay. Then, 15 μL proteins were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were then blocked with 5% free-fat milk for 1 h at room temperature and incubated with primary anti-phospho-Cx43, anti-Cx43, anti-COX IV, anti-GSTZ1, anti-CYBB, anti-ATOX1, anti-GSS, anti-HMOX1, and anti-GAPDH antibodies for 2 h at room temperature. Subsequently, membranes were incubated with horseradish peroxidase (HRP)-labeled Goat Anti-Rat IgG for 1 h at 37 °C, followed by washing 3 times with Tween-20 (TBST) and then developed using an enhanced chemiluminescence detection kit. Intensity of the bands was detected and analyzed using Quantity One analyzing system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
3
Western Blot Analysis of MECP2, DNMT Proteins
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Extracted protein was boiled in SDS/β-mercaptoethanol sample buffer, and 50 μg protein was run on 5–12% gradient polyacrylamide gel and transferred to PVDF membranes (Amersham, St Albans, Herts, UK). Membranes were incubated with rabbit anti-MECP2 polyclonal antibody (GeneTex, USA, 1:1000), mouse anti-β-ACTIN monoclonal antibody (Abcam, Cambridge, MA USA, 1:1000), rabbit anti-DNMT1, DNMT3a and DNMT3b polyclonal antibody (1:1000, SantCruz Biotechnology Inc., CA, USA) overnight at 4 °C. After that, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA, 1:10, 000) at room temperature for 1 h. ECL detection reagents (Millipore, Billerica, MA, USA) were added on the membranes for 1 min and were immediately exposed to X-ray film (Kodak, USA). The β-actin signal was used as a loading control. The experiment has been repeated at least three times. The bands were analyzed using Quantity One analyzing system (Bio-Rad, Hercules, CA, USA). The protein level was represented as the relative ratio of the MECP2, DNMT1, DNMT3a and DNMT3b signals vs the housekeeping gene (β-ACTIN).
4
SDS-PAGE and Western Blotting Protocols
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SDS-PAGE and Western Blotting (WB) were carried out according to standard protocols [50 (link), 51 (link)]. Briefly, cells were lysed and whole cells lysates (∼50 μg per lane) were separated using 4–12% Novex Bis-Tris SDS-acrylamide gels (Invitrogen) and electro-transferred on nitrocellulose membranes (Bio-Rad). Then, nitrocellulose membranes were blocked with milk and probed over-night with primary antibodies at 4°C, then membranes were washed 3 times in PBS-Tween, incubated with a secondary antibody conjugated with horseradish peroxidase for 2 hours at RT. Chemiluminescence was detected using Western Blotting Luminol Reagent (Santa Cruz, Dallas, TX, USA). Signal intensity was quantified with the Quantity One Analyzing System (Bio-Rad). Antibodies used were the following: EZH2 (cat#5246S), CDK6 (cat#3136S) and MCL1 (cat#5453S) were from Cell Signaling, while SP1 (sc-14027), ACTIN (sc-1616) and GAPDH (sc-25778) were from Santa Cruz.
5
SDS-PAGE and Western Blotting Analysis
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SDS-PAGE and Western blotting were performed according to standard protocols. Briefly, cells were homogenized in lysis buffer containing 15 mM Tris/HCl pH 7.5, 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton X-100, and Halt Protease Inhibitor Single-Use cocktail (100X, Thermo Scientific, Waltham, MA, USA). Whole cell lysates (50 µg per line) from transfected cell lines were separated using 4–12% Novex Bis-Tris SDS-acrylamide gels (Invitrogen), electro-transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and immunoblotted with the p27Kip1 (SX53G8.5) mouse mAb (Cell Signaling, Beverly, MA, USA). Membranes were washed 3 times in PBS-Tween and then incubated with a secondary antibody conjugated with horseradish peroxidase in 0.5% milk for 2 hours at room temperature. Chemiluminescence was detected using Western Blotting Luminol Reagent (sc-2048, Santa Cruz, Dallas, TX, USA). Signal intensity was quantified with the Quantity One Analyzing System (Bio-Rad).
6
Western Blot Analysis of HIF3α Protein
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Extracted protein was boiled in SDS/β-mercaptoethanol sample buffer. Sixty microgram protein was run on 6–10% gradient polyacrylamide gel and transferred to PVDF membranes (Amersham, St Albans, Herts, UK). The membranes were incubated with rabbit anti-HIF3α polyclonal antibody (GeneTex, USA, 1:1000) or mouse anti-β-ACTIN monoclonal antibody (Abcam, Cambridge, MA USA, 1:1000) 2 h at 27°C. Then the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA, 1:10,000) 1 h at 37°C. ECL The membranes were incubated with ECL reagents 1 min (Millipore, Billerica, MA, USA) and exposed to X-ray film (Kodak, USA). The β-ACTIN was used as a loading control. The experiment was repeated at least three times. The bands were analyzed using Quantity One analyzing system (Bio-Rad, Hercules, CA, USA). The protein level was represented as the relative ratio of the HIF3α signals vs. β-ACTIN signals. The detection of HIF3α protein level in HTR8/SVneo cell transfected with miR-29b mimic and inhibitor was repeated for three times (n = 3).
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