Whole cell lysates were prepared in RIPA buffer from BCL as previously described (Mostafa et al., 2014 ). Proteins were quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, #23225) and reduced with 2‐mercaptoethanol (Gibco, #21985023). RAD51C protein expression was assessed in BCL whole cell lysates by electrophoresing 25 µg of protein by 10% SDS‐PAGE, followed by western blotting. Membranes were blocked in 5% milk powder in TBS‐Tween (0.15 M NaCl, 0.05 M Tris pH 7.4, 0.05% Tween 20) for 1 hr and incubated overnight with 4 µg/ml monoclonal mouse anti‐RAD51C (2H11) antibody (Santa Cruz Biotechnology, #sc‐56214) or 250 ng/ml mouse anti‐alpha tubulin (B‐7) antibody (Santa Cruz Biotechnology, #sc‐5286) at 4°C. Antibody binding was detected with 1:10,000 dilutions of horseradish peroxidase‐conjugated goat anti‐mouse secondary antibody (Jackson Immunoresearch, #115‐036‐071) and Clarity Western ECL substrate (Bio‐Rad, #1705060). Immunoreactivity was visualized and quantified by scanning densitometry using an ImageQuant LAS 4000 and ImageQuant TL Software v8.1, respectively (GE Healthcare). Five independent experiments were performed, and RAD51C protein expression was normalized to that of alpha tubulin.
Horseradish peroxidase conjugated goat anti mouse secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in Panama, United Kingdom, United States
Horseradish peroxidase-conjugated goat anti-mouse secondary antibody is a laboratory reagent used to detect and quantify mouse primary antibodies in various immunoassays. The antibody is conjugated with the enzyme horseradish peroxidase, which can catalyze a colorimetric or chemiluminescent reaction for signal detection.
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Lab products found in correlation
Nupage sds page system, by Thermo Fisher Scientific (2 mentions)
Amersham hyperfilm ecl film, by GE Healthcare (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (2 mentions)
Whatman, by Cytiva (2 mentions)
Imagequant tl software v8, by GE Healthcare (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Las 4000, by Cytiva (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Protran, by Cytiva (1 mentions)
Anti gpx1, by Abcam (1 mentions)
Senescence associated β galactosidase sa β gal staining kit, by Cell Signaling Technology (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh2 da, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
8 protocols using horseradish peroxidase conjugated goat anti mouse secondary antibody
1
Quantifying RAD51C Protein Expression
2
Western Blot Analysis of Fly Heads
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10 adult fly heads/sample were prepared in sample buffer using standard methods. SDS-PAGE was performed using the Novex NuPAGE SDS-PAGE system with 4%-12% Bis-Tris gels run at 125 V for 10 minutes and 150 V for 2.5 hours. Transfer to PVDF membrane (Bio-Rad, Hercules, CA) was performed using a Trans-Blot-SDSemi-Dry Transfer Cell (Bio-Rad, Hercules, CA). Blocking was performed in 5% BSA for GFP blots or 5% milk for actin blots in 1X PBS with 0.1% Tween 20. Primary antibodies were obtained from the DSHB, mouse anti-actin (JLA20) 1:1000, or from Torrey Pines Biolabs, rabbit anti-GFP 1:2000. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 1:5000 for actin blots. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 1:5000 for GFP blots. All antibodies were diluted in blocking buffer. Blots were developed with Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) and imaged with Amersham Hyperfilm ECL film (GE Healthcare Limited, Buckinghamshire, UK). Band intensity was quantified using ImageJ.
3
Western Blot Analysis of P-glycoprotein
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Total protein lysates were extracted and Western blot analysis was performed as previously described [88 (link)]. Following extraction, protein concentration was determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Proteins were resolved on 7% polyacrylamide gels and blotted onto a Protran BA83 cellulose nitrate membrane (Whatman, GE, Maidstone, UK), and reacted with an anti-P-gp monoclonal antibody (JSB-1, kindly provided by Dr. G. Scheffer, VU Medical Center, Amsterdam, The Netherlands). The membrane was then reacted with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch Labs, West Grove, PA). The membrane was stripped off and finally reacted with an anti-α-tubulin antibody for evaluation of actual loading (Sigma Aldrich, St. Louis, MO, USA). Quantification of the protein bands was performed using the EZ-Quant software (EZ-Quant, Tel-Aviv, Israel).
4
UVB-induced Skin Aging: Mechanisms and Mitigation
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Dulbecco's modified Eagle's medium (DMEM), penicillin, and streptomycin were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from GE Healthcare Life Sciences (Logan, UT, USA). UVB light (Philips 311 nm, TL 20W/01) was from Philips Lighting Holding B.V. (Eindhoven, The Netherlands). Cell Counting Kit-8 (CCK-8) was from Beyotime Institute of Biotechnology. RNase A, propidium iodide, FITC-phalloidin, and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Senescence-associated β-galactosidase (SA-β-gal) staining kit (#9860) was from Cell Signaling Technology Inc. (Danvers, MA, USA); terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP-biotin nick end labeling (TUNEL) staining kit was from Roche Molecular Biochemicals (Mannheim, Germany). Anti-CD31 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse antibody was from Dako (Glostrup, Denmark). Anti-GPX-1 (#ab108427), anti-catalase (#ab16731), anti-SOD-2 (#ab13533), anti-SOD-1 (#ab13498), anti-COL-1 (#ab6308), and β-actin (#ab6276) were from Abcam (Cambridge, UK); horseradish peroxidase-conjugated goat anti-mouse secondary antibody (#115-035-062) was from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).
5
SDS-PAGE Analysis of Fasciclin II
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SDS-PAGE was performed using the Novex NuPAGE SDS-PAGE system with 4%-12% Bis-Tris gels run at 125 V for 5 hours to achieve separation of individual FasII bands. Transfer to nitrocellulose membrane (Whatman, Dassel, Germany) was performed using a Trans-Blot-SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA). Blocking was performed in 5% BSA for FasII blots or 5% milk for actin blots in 1X PBS with 0.1% Tween 20. Primary antibodies were obtained from the DSHB: mouse anti-FasII (1D4) 1:100 and mouse anti-actin (JLA20) 1:1000. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 1:1000 for FasII blots and 1:5000 for actin blots. All antibodies were diluted in blocking buffer. Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) and imaged with Amersham Hyperfilm ECL film (GE Healthcare Limited, Buckinghamshire, UK). Band intensity was quantified using ImageJ.
6
HER2 Immunohistochemistry in Breast Cancer
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Immunohistochemistry was performed according to previously described methods using a normal light microscope (magnification, ×400) (28 (link)). Paraffin-embedded tissues from surgery were deparaffinized and pretreated in a microwave. The slides were incubated using monoclonal mouse anti-HER2 antibodies (1:200, Proteintech 60311-1-lg, China) for 1 h at 37°C. After rinsing with PBS (pH 7.4), sections were treated with a horseradish peroxidase conjugated-goat-anti-mouse secondary antibody (1:2,000, Jackson ImmunoResearch, Inc., West Grove, PA, USA; cat no. 115-035-003) at room temperature for 1 h. Then, the slides were incubated with 3-diaminobenzidine solution for 20 min at room temperature. Patients with HER2+ breast cancer was diagnosed by FISH in the Second Affiliated Hospital of Soochow University as described previously (22 (link)).
7
Adenovirus Vaccine Expressing GUCY2C-S1
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Adenovirus containing mouse extracellular domain (GUCY2C1-429) with the influenza HA107-119 CD4+ T-cell epitope known as site 1 (S1) was described previously (Ad5-GUCY2C-S1).20 (link) Here, GUCY2C-S1 was cloned into pShuttle and subcloned into the E1 region of previously generated replication-deficient chimeric adenovirus (Ad5.F35) in which the Ad5 fiber was replaced by the Ad35 fiber22 (link) to generate Ad5.F35-GUCY2C-S1. All adenovirus vaccines used in this study were produced in HEK293 cells and purified by cesium chloride ultracentrifugation under Good Laboratory Practices by the Baylor College of Medicine in the Cell and Gene Therapy Vector Development Lab and certified to be negative for replication-competent adenovirus, mycoplasma, and host cell DNA contamination. In vitro GUCY2C-expression experiments (dose–response and time–course) were carried out in A549 (American Type Culture Collection (ATCC)) cells. Virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points. Relative GUCY2C levels were quantified in supernatants by western blot using 2 μg/mL MS7 mouse anti-GUCY2C monoclonal antibody23–25 (link) and 0.1 μg/mL horseradish peroxidase-conjugated goat antimouse secondary antibody (Jackson Immuno).
8
Western Blot Analysis of scFv
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Proteins mixed with 2× SDS-PAGE loading buffer were separated by electrophoresis on a SDS-12.5% PAGE, and electro-transferred onto a polyvinylidene fluoride membrane in the Towbin buffer (25 mM Tris base, 192 mM glycine, pH 8.3, 0.1% SDS, and 20% methanol) at 100 V for 90 min. After blocking the membrane with TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) containing 5% skim milk, scFv was detected by sequential incubations with 1000-fold diluted mouse monoclonal Ab against hexahistidine tag (Proteintech, catalog no. 66005-1-Ig) and 5000-fold diluted horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories), followed by reaction with chemiluminescent substrates (PerkinElmer).
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