Total proteins were extracted from the rat myocardial tissues and H9C2 cells using protein lysis buffer (Thermo Fisher Scientific, Inc.) with a protease inhibitor (Thermo Fisher Scientific, Inc). Protein concentration was measured by a BCA kit (Pierce, Rockford, IL) according to the manufacturer's instructions. 40 μg of protein extracts were separated using 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham Biosciences, Bucks, United Kingdom). After blocking with 5% BSA in 0.5% Tween 20 in phosphate-buffered saline at 4°C overnight, the membranes were incubated with anti-BRD4 polyclonal antibody (1:500; cat. no. ab75898; Abcam, Cambridge, MA), anti-GAPDH monoclonal antibody (1:2000; cat. no. ab181602; Abcam), anti-phospho-p85 polyclonal antibody (1:500; cat. no. ab182651; Abcam), anti-P85 polyclonal antibody (1:500; cat. no. 4292; Cell Signaling Technology, Beverly, MA), anti-AKT 1/2 monoclonal antibody (1:800; cat. no. ab182729; Abcam), anti-AKT (phospho S473) monoclonal antibody (1:400; cat. no. ab81283; Abcam), and anti-AKT (phospho T308) monoclonal antibody (1:400; cat. no. ab38449; Abcam) at 37°C for 2 hours. Membranes were washed with TBST and incubated with secondary antibody labeled with horseradish peroxidase (HRP; 1:1000; cat. no. ab97051; Abcam) at 37°C for 1 hour, followed by an enhanced chemiluminescence detection kit (Amersham Biosciences).
Anti gapdh monoclonal antibody
Manufactured by Abcam
Sourced in United States
Anti-GAPDH monoclonal antibody is a laboratory reagent used to detect the presence and quantity of the GAPDH protein in biological samples. GAPDH is a common housekeeping gene, and its protein product is involved in glycolysis. This antibody can be used in various immunodetection techniques, such as Western blotting, to identify and quantify GAPDH expression levels in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Protein lysis buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Block it alexa fluro red fluorescent control, by Thermo Fisher Scientific (1 mentions)
Silencer select gapdh positive control, by Thermo Fisher Scientific (1 mentions)
Wp1066, by Merck Group (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting analysis system, by GE Healthcare (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti ubiquitin, by Abcam (1 mentions)
Anti itch, by BD (1 mentions)
Anti txnip, by Abcam (1 mentions)
Anti vlcad, by Abcam (1 mentions)
10 protocols using anti gapdh monoclonal antibody
1
Protein Extraction and Western Blot Analysis
2
Regulation of STAT3 and FOXL2 in Cells
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Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Sequence-specific Silencer Select siRNAs targeting human STAT3 mRNA (Catalogue #4390824/s743, sense: 5′-GCCUCAAGAUUGACCUAGATT − 3′, antisense: 5′-UCUAGGUCAAUCUUGAGGCCT-3′) or FOXL2 mRNA (Catalogue #4392420/s2068, sense: 5′-CGAAGUUCCCGUUCUACGATT-3′, antisense: 5′-UCGUAGAACGGGAACUUCGCG-3′) were purchased from Thermo Fisher Scientific (Waltham, USA). The scrambled Silencer Select Negative Control #1 siRNA (Catalogue #4390843), Silencer Select GAPDH Positive Control (Catalogue #4390849) and BLOCK-IT Alexa Fluro Red Fluorescent Control (Catalogue #14750100), both purchased from Thermo Fisher Scientific (Waltham, USA). Anti-STAT3 (phospho Y705) monoclonal antibody (Catalogue #ab76315), anti-FOXL2 monoclonal antibody (Catalogue #ab188584) and anti-GAPDH monoclonal antibody (Catalogue #ab181602) were all purchased from Abcam (Cambridge, MA, USA). Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.
3
Western Blot Analysis of EpCAM Protein
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Seventy two hours after transfection, cells were lysed with RIPA lysis buffer (Beyotime, Beijing, China) for 30 min on ice. Protein was electrophoresed on 10%SDS-PAGE and blotted to PVDF membranes. Membranes were blocked in TBST buffer with 5% fat-free milk and incubated separately with primary anti-EpCAM monoclonal antibody (1:1000, Cell Signaling Technology, USA) or anti-GAPDH monoclonal antibody (1:1000, Abcam, USA) at 4°C. After overnight incubation and washing, membranes were incubated with secondary antibody (1:5000, Zhongshan Goldenbridge, China) at room temperature for 2 h. Western Chemiluminescent HRP Substrate (Millipore, USA) was used to visualizing the immunoblots.
4
Prostate Cancer Cell Apoptosis Assay
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Rabbit anti-human GRP78 polyclonal antibody, mouse anti-human CHOP monoclonal antibody, mouse anti-human hnRNPA1 monoclonal antibody, mouse anti-human cleaved caspase-3 monoclonal antibody, and anti-GAPDH monoclonal antibody were purchased from Abcam. Que and PTX were purchased from Dalian Meilun Biotechnology Co., Ltd. TRIzol® reagent was purchased from Invitrogen (Gibco, Shanghai, China), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Shanghai, China).
Human prostate cancer (PC-3) cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Paisley, UK) containing 10% fetal bovine serum (FBS) at 37°C in a humidified environment containing 5% CO2.
Nude male BALB/c mice (4–6 weeks old, weighing 14–21 g) were purchased from Jining Medical University (Jining, China). All the animal procedures were performed in accordance with the National Institute of Health guidelines and were approved by the Jining First People’s Hospital of Jining Medical University (Medical ethics committee of the Jining First People’s Hospital of Jining Medical University).
Human prostate cancer (PC-3) cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Paisley, UK) containing 10% fetal bovine serum (FBS) at 37°C in a humidified environment containing 5% CO2.
Nude male BALB/c mice (4–6 weeks old, weighing 14–21 g) were purchased from Jining Medical University (Jining, China). All the animal procedures were performed in accordance with the National Institute of Health guidelines and were approved by the Jining First People’s Hospital of Jining Medical University (Medical ethics committee of the Jining First People’s Hospital of Jining Medical University).
5
Western Blot Analysis of NAD-IDH Protein
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Western blot analysis was performed as previously reported34 (link). Briefly, the cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA), and the proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-P (Millipore, Billerica, MA, USA). The membranes were probed with an anti-NAD-IDH (IDH3) α polyclonal antibody (1:1,000; Abcam, Cambridge, UK), an anti-β-actin monoclonal antibody (1:5,000; Sigma-Aldrich), and an anti-GAPDH monoclonal antibody (1:2,000; Abcam). The membranes were then incubated with secondary antibodies against rabbit or mouse IgG conjugated to horseradish peroxidase (Cell Signaling Technology). The bands were visualized using the ECL Western Blotting Analysis System (GE Healthcare, Buckinghamshire, UK), and the images were acquired using a LAS-3000 Imager (FUJIFILM UK Ltd., Systems, Bedford, UK). The density of each band was quantified using the ImageJ software (NIH, Bethesda, MD, USA).
6
Quantifying Protein Expression in Fibroblasts
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Western blotting of whole cell lysates from patient and control fibroblasts was performed as previously described [20 ]. Twenty five micrograms of protein were loaded onto a 4–15% gradient precast SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA). Following electrophoresis separated proteins were transferred onto a nitrocellulose membrane. Primary antibodies included anti-ITCH (1;1000, BD Transduction Laboratories, San Jose, CA, #611198), Anti-VLCAD (1:1000, antigen produced by the Vockley Lab, UPMC Children's Hospital of Pittsburgh and antibody generated by Cocalico Biologics, Stevens, PA), Anti-TFP α/β (1:2000, produced by the Vockley Lab), anti-Ubiquitin (1:1000, Abcam, Cambridge, MA, #ab19247), and anti-TXNIP (1:500, Abcam, #ab188865). Blots were incubated with HRP conjugated secondary antibodies. Primary purified mouse anti-GAPDH monoclonal antibody (1,25,000, Abcam, #ab8245) was used as a loading control (Supplemental Table S1). All western blot studies were performed more than once from different celluar lysates (technical replicates).
7
Protein Fractionation and Western Blot Analysis
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Cytoplasmic and nuclear protein fractions were isolated from NSCs by means of NE-PER Extraction Kit (Thermo Fisher Scientific). Total extracts from SVZ and hippocampal regions of control mice were lysed in RIPA buffer solution (Pierce, Rockford, IL, USA) containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 1% TX-100, 0.1% sodium dodecyl sulfate (SDS) and ×1protease inhibitor mixture (Sigma), 1 mM sodium orthovanadate, and 1 mM NaF. All samples were resolved by Western blot standard procedures61 (link)–63 (link), using the following antibodies: polyclonal anti-β-Catenin antibody (1:4000, Abcam, Cat. #ab6302), monoclonal anti-actin antibody (1:4000, Sigma, Cat. #T6074), monoclonal anti-GAPDH antibody (1:2000, Abcam, Cat. #ab8245), monoclonal anti-fibrillarin (1:1000, Thermo Scientific, Cat. #MA3-16771). All experiments were repeated independently 3 times.
8
Western Blot Analysis of ATF2 and GAPDH
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Total proteins were extracted using RIPA buffer (Pierce, Rockford, IL, USA). The protein concentration was determined using Bradford method (Pierce). Equal amounts of proteins (40 μg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in PBS-Tween-20 for 1 h, the membranes were incubated at 4°C overnight with anti-ATF2 antibody (1 : 500 dilution, Abcam, Hong Kong, China) or monoclonal anti-GAPDH antibody (1 : 5000, Abcam). After washing with Tris-buffered saline and Tween 20, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1 : 5000, Abcam) at 37°C for 1 h. The immunohistochemical reaction was detected using an ECL Plus Detection kit (Pierce).
9
Western Blot Analysis of REST, DHCR24 and GAPDH
10
Protein Expression Analysis in Cells
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A total of 5 × 105 cells/well for each treatment were pelleted and lysed in RIPA lysis buffer supplemented with protease inhibitor. Cell lysates were centrifuged at 12,000× g for 15 min at 4 °C, and protein concentration was measured using the Bradford method (Bio-Rad). Proteins were separated on SDS-PAGE in reducing conditions and transferred to nitrocellulose membrane (Bio-Rad). After saturation with 5% milk, the membrane was incubated overnight at 4 °C with the appropriate primary antibody at the indicated dilutions—anti-COX-2 (Abcam—diluted 1:3000) and anti- iNOS (Abcam—diluted 1:2000)—washed, incubated with HRP-conjugated secondary antibody for 1 h at room temperature, and revealed using the ECL substrate (Bio-Rad). Monoclonal anti-GAPDH antibody (Abcam—diluted 1:10,000) was used for protein loading control. Densitometry analysis was performed using ImageJ.
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