Cells were incubated for 5 min in lysis buffer (Complete lysis M; Roche Diagnostics, Basel, Switzerland). The protein concentration in the lysate was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein (20 μg) were dissolved in NuPAGE LDS sample buffer (Invitrogen) and a 10% sample reducing agent (Invitrogen). The lysates were boiled at 70 °C for 10 min and then loaded into and subjected to electrophoresis in NuPAGE 4–12% Bis-Tris gels (Invitrogen) at 200 V for 60 min. The proteins were then transferred onto polyvinylidene difluoride membranes (Invitrogen), and the membranes were blocked with WesternBreeze Blocker/Diluent (Invitrogen). The membranes were then probed with the anti-FLG antibody, anti-OVOL1 antibody, and a mouse monoclonal antibody against human β-actin (anti-β-actin) (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse IgG antibodies (Cell Signaling Technology) served as a secondary antibody. The visualization of protein bands was accomplished with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) by ChemiDoc touch imaging system (Bio-Rad).
Horseradish peroxidase conjugated anti mouse igg antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated anti-mouse IgG antibody is a secondary antibody conjugated with the enzyme horseradish peroxidase. It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Westernbreeze blocker diluent, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Complete lysis m, by Roche (1 mentions)
Lysis buffer, by Roche (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mouse monoclonal anti mt 1 2 antibody e9, by Agilent Technologies (1 mentions)
May gr nwald and giemsa stain solution, by Merck Group (1 mentions)
Cmf pbs, by Nissui Pharmaceutical (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
7 protocols using horseradish peroxidase conjugated anti mouse igg antibody
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Phospho-Stat6
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Cells were incubated for 5 min in lysis buffer (Roche Diagnostics, Basel, Switzerland). The protein concentration in the lysate was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein (20 μg) were dissolved in NuPAGE LDS sample buffer (Invitrogen) and a 10% sample reducing agent (Invitrogen). The lysates were boiled at 70 °C for 10 min and then loaded into and subjected to electrophoresis in NuPAGE 4%–12% Bis-Tris gels (Invitrogen) at 200 V for 60 min. The proteins were then transferred onto polyvinylidene difluoride membranes (Invitrogen), which were blocked with WesternBreeze Blocker/Diluent (Invitrogen). The membranes were then probed with anti-phosphorylated Stat6 rabbit polyclonal antibody (tyrosine 641) (Abcam, Cambridge, UK), anti-Stat6 rabbit polyclonal antibody (Abcam) and anti-histone H3 rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse IgG antibodies (Cell Signaling Technology) served as secondary antibodies. The visualization of protein bands was accomplished with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) using the ChemiDoc touch imaging system (Bio-Rad).
3
Dioscin Regulates Autophagy in Cells
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Dioscin was kindly provided by Professor Jinyong Peng (College of Pharmacy, Dalian Medical University, Dalian, China). The purity of dioscin was 96.55% as determined by HPLC55 (link). ADR was from Shenzhen Main Luck Pharmaceuticals, Inc. (Shenzhen, China). DMEM was purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Invitrogen Life Technologies Corporation (Invitrogen, Carlsband, CA, USA). TNF-α was purchased from Peprotech (Rocky Hill, NJ, USA). 3-MA, LY294002, antibodies against LC3-I/II, Akt, Phospho-Akt, PI3K, Phospho-PI3K phospho-IκB-α and horseradish peroxidase-conjugated anti-mouse IgG antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against β-actin and MDR1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was from USB Corporation (Cleveland, OH, USA). All other reagents and solvents were of analytical grade.
4
ZIKV Protein Characterization Protocol
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The culture medium of Vero E6 cells infected with each ZIKV were overlaid on 20% sucrose and ultracentrifuged at 28,000 × g at 4°C for 2 hrs, and viruses were recovered from the pellet. The proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with 15% polyacrylamide gels at 100 V for 2 hrs and transferred onto Immobilon-P membrane (Millipore Corp, Bedford, MA) at 100 V for 1 hr. Membranes were blocked with 3% skim milk in PBS for 2 hrs at room temperature followed by the addition of diluted hyperimmune mouse sera against ZIKV. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody (Cell Signaling Technology, Danvers, MA). To produce hyperimmune sera against ZIKV, IFNAR-/- mice were initially infected s.c. with 1 × 104 CCID50 of Natal RGN strain (GenBank accession number: KU527068), and 3 weeks later, the mice were infected s.c. with 1 × 103 CCID50 of MR766 twice more separated by a 3-week interval. The serum samples were collected 4 days after the second MR766 infection. The bound antibodies onto the membrane were visualized with chemiluminescence (Clarity Western ECL Substrate, Bio-Rad). Band densities were determined by using ImageJ software (US National Institutes of Health, Bethesda, MA).
5
Bovine Aortic Endothelial Cell Culture
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Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the respective vendors: Dulbecco's modified Eagle's medium (DMEM) and calcium-and magnesium-free phosphate-buffered saline (CMF-PBS; Nissui Pharmaceutical, Tokyo, Japan); fetal bovine serum and a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA); QIAzol lysis reagent (QIAGEN, Valencia, CA, USA); GeneAce SYBR qPCR Mixα (Nippon Gene, Tokyo, Japan); mouse monoclonal anti-MT-1/2 antibody (E9; Dako, Glostrup, Denmark); Mouse monoclonal anti-β-actin antibody (Wako Pure Chemical Industries, Osaka, Japan); horseradish peroxidase-conjugated antimouse IgG antibody (#7076; Cell Signaling, Beverly, MA, USA); May-Grünwald and Giemsa stain solution (Merck KGaA, Darmstadt, Germany); and cadmium chloride, manganese chloride tetrahydrate, and other reagents (Nacalai Tesque, Kyoto, Japan).
6
Western Blot Analysis of Myo1C Protein
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Proteins were extracted from the mononuclear cells of human peripheric blood with lysis buffer (20 mM Tris-HCl, pH 8.0, 1% Triton-X100, 150 mM NaCl, 50 mM NaF, 0.1% SDS) containing 1 mM PMSF and 10 μg/ml of cocktail of protease inhibitors “Complete” (Roche, Basel, Switzerland), subjected to electrophoresis in 12%–15% SDS-PAGE, and transferred onto the nitrocellulose membrane (Schleicher and Schuell, Germany). The membrane was blocked with 3% BSA for 1 h at 23 °C, washed with the PBST buffer (PBS supplemented with Tween-20) (three times for 5 min of each wash), and incubated overnight with primary human antibody (Myo1C, AVIVA SYSTEM BIOLOGY, No ARP56292), 0.1 μg/ml in 5% bovine serum albumin (BSA)-supplemented with the PBST. After incubation, the blotted membrane was washed with the PBST (three times for 5 min each wash), and incubated for 1.5 h with horseradish peroxidase-conjugated anti-mouse IgG antibody (Cell Signaling Technology Europe , the Netherlands). Proteins on the blot were visualized with the ECL reagent (Sigma, USA).
7
Western Blot Analysis of ChAT Expression
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HEK293T or ChAT‐HEK293 cells were washed twice with D‐PBS (−) and directly lysed with 1 × SDS/PAGE sample buffer (62.5 mm Tris/HCl, 3 w/v% SDS, 7.5% glycerol, 0.005 w/v% bromophenol blue, and 50 mm dithiothreitol). All samples were run on e‐PAGEL (ATTO, Tokyo, Japan) and then transferred onto a polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim milk in Tris‐buffered saline containing 0.1% Tween‐20, membranes were processed through sequential incubations with goat anti‐ChAT (working dilution 1 : 1000; Chemicon, Temecula, CA, USA) overnight and then with a horseradish peroxidase‐conjugated anti‐goat IgG antibody (working dilution 1 : 10,000, Jackson Laboratory, Bar Harbor, ME, USA) for 2 h. An internal standard, β‐actin, was detected using anti‐β‐actin antibodies followed by a horseradish peroxidase‐conjugated anti‐mouse IgG antibody (working dilution 1 : 3000, Cell Signaling Technology, Danvers, MA, USA). Bound antibodies were visualized using a chemiluminescence imager (Amersham, Little Chalfont, UK), detected with Light‐Capture (AE‐6972; ATTO), and quantified using CS Analyzer (ver. 3.00 software; ATTO).
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