Antibodies for western blotting were as follows: the GAPDH antibody (cat. no. ab8245) was purchased from Abcam, and the Bcl-2 (cat. no. 3498), pro-caspase 3 (cat. no. 9662), cleaved caspase 3 (cat. no. 9661), phosphorylated (p)-Akt (S473) (cat. no. 9271) and Akt antibodies (cat. no. 9272) were purchased from Cell Signaling Technology, Inc. Goat anti-mouse secondary antibody (cat. no. 31430) and goat anti-rabbit secondary antibody (cat. no. 31460, both at 1:20,000 dilution) were from Thermo Fisher Scientific, Inc. HDN (HPLC>98%; cat. no. XW05202631) was obtained from Sinopharm Chemical Reagent Co., Ltd. LY294002 (cat. no. L9908) was purchased from Sigma-Aldrich (Merck KGaA). Malondialdehyde assay kit (MDA; cat. no. A003-1), superoxide dismutase assay kit (SOD; cat. no. A001-1), catalase assay kit (CAT; cat. no. A007-2), glutathione peroxidase assay kit (GPx; cat. no. A006-1) and glutathione assay kit (GSH; cat. no. A006-1) were purchased from Nanjing Jiancheng Bioengineering Institute.
Goat anti mouse secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Goat anti-mouse secondary antibody is a laboratory reagent used to detect and visualize mouse primary antibodies in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. It is a polyclonal antibody produced in goats that specifically binds to the Fc region of mouse immunoglobulins, allowing for the indirect detection of mouse target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit secondary antibody, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Bcl 2, by Abcam (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ecl plus western blotting detection reagent, by Cytiva (1 mentions)
Gradient sds page, by Bio-Rad (1 mentions)
Ab226943, by Abcam (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Annexin apoptosis assay kit, by Beyotime (1 mentions)
Transwell chamber, by BD (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Chemiluminescence system, by PerkinElmer (1 mentions)
Cdc34, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
14 protocols using goat anti mouse secondary antibody
1
Western Blot Analysis of Apoptosis Markers
2
Quantitative Protein Analysis of Influenza A M2, Actin, and HO-1
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Total proteins were extracted by ice-cold M-PER mammalian protein extraction reagent containing halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentrations were determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amount of samples (20 μg protein) were subjected to SDS-PAGE using a 10% (w/v) running gel, and then electro-transferred to PVDF membrane (Millipore, Billerica, MA, USA) and blocked by 5% (w/v) milk for 1 h at room temperature. The membranes were incubated with mouse antibodies against influenza A M2 (Santa Cruz, USA), actin (Cell Signaling Technology, USA) and HO-1 (Abcam Cambridg, USA) for 2 h at room temperature. Then goat-anti-mouse secondary antibody (Cell Signaling Technology, USA) was incubated for 1 h at room temperature. The signals were detected using ECL detection kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
3
Western Blot Analysis of Inflammatory Signaling
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Whole cell lysates from infected, differentiated THP-1 cells were obtained by the addition of lysis buffer (20 mM HEPES, 1.0% [v/v] Triton X-100, 0.1% [w/v] SDS, 150 mM NaCl, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride) and subsequent incubation at 4°C. Cell lysates were separated by electrophoresis in a denaturing 12% SDS-PAGE, and subsequent transfer to PVDF membrane. Blots were incubated overnight with relevant primary antibodies at 4°C, washed 3 times with PBS, and then incubated with appropriate HRP-conjugated, secondary antibody (Jackson Immunochemicals). Blots were developed following incubation in ECL Plus Western Blotting Detection Reagent (Amersham Bioscience). Antibodies against phospho-p65 (serine 536), total p65, and total p38 were obtained from Cell Signaling. Rabbit polyclonal antibody against phospho-p38 was obtained from Promega.
For analysis of flagellin expression by S. Typhimurium I77 and D65, bacterial lysate and supernatant were separated on 4–15% gradient SDS-PAGE (Biorad) and transferred to PVDF membrane. The blot was incubated overnight in a 1∶1000 dilution of FliC monoclonal S. Typhimurium (IE6) AH12 antibody and a goat-anti-mouse secondary antibody (Cell Signaling). Densitometry analysis of the bands was performed using the ImageJ software (http://imagej.nih.gov/ij/ ).
For analysis of flagellin expression by S. Typhimurium I77 and D65, bacterial lysate and supernatant were separated on 4–15% gradient SDS-PAGE (Biorad) and transferred to PVDF membrane. The blot was incubated overnight in a 1∶1000 dilution of FliC monoclonal S. Typhimurium (IE6) AH12 antibody and a goat-anti-mouse secondary antibody (Cell Signaling). Densitometry analysis of the bands was performed using the ImageJ software (
4
Western Blot Analysis of Fusion Proteins
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The fusion proteins were stained with anti-human CD9 antibody (produced in mouse, 60232-1-lg, Proteintech, Rosemont, IL, USA), or anti-human AGO2 antibody (produced in rabbit, ab226943, Abcam, Boston, MA, USA).
The membrane was incubated with 1:1000 diluted primary antibody overnight at 4 °C and then with 1:500 diluted horseradish peroxidase conjugated goat anti-mouse secondary antibody (7076, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit secondary antibody (7074, Cell Signaling Technology) for 2 h at room temperature. Antibody dilutions were made in Immobilon® Block—CH solution (Chemiluminescent Blocker, Merck Millipore, Darmstadt, Germany). After each incubation step, membranes were washed three times in the PBS containing 0.2% Tween-20 for 5 min. Protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific™) with ChemiDoc XRS System (Bio-Rad Laboratories).
The membrane was incubated with 1:1000 diluted primary antibody overnight at 4 °C and then with 1:500 diluted horseradish peroxidase conjugated goat anti-mouse secondary antibody (7076, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit secondary antibody (7074, Cell Signaling Technology) for 2 h at room temperature. Antibody dilutions were made in Immobilon® Block—CH solution (Chemiluminescent Blocker, Merck Millipore, Darmstadt, Germany). After each incubation step, membranes were washed three times in the PBS containing 0.2% Tween-20 for 5 min. Protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific™) with ChemiDoc XRS System (Bio-Rad Laboratories).
5
Skp2-Mediated Cell Cycle Regulation
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PF was purchased from the HuanYu Biotechnology Development Company. MTT was purchased from Sigma-Aldrich; Merck KGaA. The Annexin apoptosis assay kit was obtained from Beyotime Institute of Biotechnology. The Transwell chambers and Matrigel were purchased from BD Biosciences. Lipofectamine® 2,000 reagent was obtained from Invitrogen; Thermo Fisher Scientific, Inc. The anti-Skp2 (1:2,000; cat. no. 4358), anti-tubulin (1:5,000; cat. no. 2146), anti-β-actin (1:5,000; cat. no. 3700), anti-cyclin dependent kinase inhibitor 1A (p21; 1:1,000; cat. no. 2491) and anti-cyclin dependent kinase inhibitor 1C (p57; 1:1,000; cat. no. 2557) antibodies were obtained from Cell Signaling Technology, Inc. Goat anti-mouse secondary antibody (1:5,000; cat. no. A-11031) and goat anti-rabbit secondary antibody (1:5,000; cat. no. A-11034) were purchased from Thermo Fisher Scientific, Inc.
6
Western Blot Analysis of Cell Cycle Regulators
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After treatment, cells were collected and lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.0% NP-40, 0.1% SDS, and 0.5% deoxycholic acid) supplemented with protease and phosphatase inhibitors. Lysates were resolved in 10% SDS-PAGE gels for western blotting. Proteins were transferred to PVDF membrane; after blocking with 5% non-fat milk, membranes were incubated with primary antibodies: Cdc34 (Santa Cruz, sc-28381, 1:1000 dilution), p27 (BD Biosciences, 610242, 1:1000 dilution), p27 (Cell Signaling, 3686, 1:1000 dilution), SKP2 (Santa Cruz, sc-7164, 1:1000 dilution), SKP1 (BD Biosciences, 610530, 1:1000 dilution), and β-actin (Sigma, A5316, 1:50,000 dilution). Thereafter, membranes were washed and incubated with relative secondary antibodies: goat anti-rabbit secondary antibody (Cell Signaling, 7074, 1:5000 dilution), or goat anti-mouse secondary antibody (Cell Signaling, 7076, 1:5000 dilution).
Finally, the signals were visualized by the chemiluminescence system (Perkin Elmer). These experiments were conducted three times. Western blot band quantification was performed using Quantity One (BioRad Laboratories, Inc.) and signals were normalized to the control group. Unprocessed scans of blots are provided in the Source Data file.
Finally, the signals were visualized by the chemiluminescence system (Perkin Elmer). These experiments were conducted three times. Western blot band quantification was performed using Quantity One (BioRad Laboratories, Inc.) and signals were normalized to the control group. Unprocessed scans of blots are provided in the Source Data file.
7
Protein Expression Analysis in Cells
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The cells were lysed in RIPA lysis buffer (Servicebio, China) containing 1% protease inhibitor for 30 minutes, and the protein supernatant was obtained after centrifugation. The supernatant was then mixed with 5× protein loading buffer (Coolaber, China) and heated at 100°C for 10 minutes in preparation for sample loading. Electrophoresis was carried out by SDS-PAGE and the proteins were then moved to NC membrane. The membranes were blocked with TBST buffer containing 5% skimmed milk powder for 2 hours and then placed in a 4°C shaking table overnight with various primary antibodies. The next day, the membrane was incubated with secondary antibody at room temperature for one hour. A Clinx Gel Documentation and Analysis instrument was used to observe the protein bands, and ImageJ was used to analyze the gray value of the bands. The antibodies used were as follows: anti-cyclin D1 (Cell Signaling Technology, USA, 1:1000), anti-CDK4 (Cell Signaling Technology, USA, 1:1000), anti-CDK6 (Cell Signaling Technology, USA, 1:1000), anti-vinculin (Abmart, China, 1:1000), anti-β-tubulin (Cell Signaling Technology, USA, 1:1000), goat anti-rabbit secondary antibody (Cell Signaling Technology, USA, 1:5000), and goat anti-mouse secondary antibody (Cell Signaling Technology, USA, 1:5000).
8
Western Blotting of Cellular Proteins
9
Visualizing Nuclear β-catenin Translocation
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Cells grown to 50% confluence on coverslips in 12-well plates were fixed with 4% paraformaldehyde solution (Biosharp, China), followed by permeabilization using 0.5% Triton X-100 (Biofroxx, German). Ensuing steps included blocking in 3% bovine serum albumin (BSA, Biofroxx, German) and overnight incubation at 4℃ with primary antibodies against β-catenin (1:200, BD Biosciences, USA). The samples were then incubated with goat anti-mouse secondary antibody conjugated to Alexa Fluor® 488 (Cell Signaling Technology (CST), USA). In another experiment, the fixed and permeabilized cells encountered YF®594-Phalloidin (UElandy, China). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime, China). The coverslips were mounted on glass slides using a protective antifade mounting medium. Samples were observed using confocal microscope (Olympus, Japan) at 600× magnification, particularly emphasizing the nuclear translocation of β-catenin, or marking the subcellular actin.
10
Protein Expression Analysis of Mutant JAG1
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NIH-3T3 cells were seeded onto a 6-well plate (Corning, NY, USA) and transfected with 3000 ng WT plasmid, mutant JAG1 plasmids or empty vectors using Lipofectamine 3000 (Invitrogen, CA, USA) when cell confluence reached approximately 70%. After 48 hours, cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer (2% SDS and 62.5 mM Tris-HCl, pH 6.8, containing protease inhibitor cocktail tablets ordered from Roche Inc.) and sonicated three times for 5 seconds. Equal amounts of protein (20 μg) were loaded onto a 10% polyacrylamide gel and analyzed by immunoblotting. The antibodies used for WB were FLAG (Cat# F1804, Sigma, St. Louis, MO, USA, 1:3000 dilution), JAG1 (Cat# 70109S, Cell Signalling Technology, Danvers, MA, USA, 1:2000 dilution) and GAPDH (Cat# 60004-1-Ig, Proteintech, Wuhan, China, 1:3000 dilution). HRP-conjugated goat antirabbit secondary antibody (Cat# 7074, Cell Signaling Technology, 1:5000 dilution) and goat antimouse secondary antibody (Cat# 7076, Cell Signaling Technology, 1:5000 dilution) were used for immunoblotting.
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