1 x 106 HeLa cells were infected at a MOI of 5. Cells and supernant were collected at 4 hpi and were lysed on ice for 5 min in RIPA lysis buffer supplemented with 1x Halt protease and phosphatase inhibitor cocktail. Cells were further processed for subcellular fractionation using a Subcellular Protein Fractionation Kit (ThermoFisher). Samples were mixed with Laemmli sample buffer (Bio-Rad), incubated at 95°C for 5 minutes, separated by SDS-PAGE, and transferred to PVDF membranes. Immunoblotting was performed using rabbit polyclonal anti-mCherry and rabbit monoclonal anti-EGFR (Abcam) followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signalling).
Horseradish peroxidase hrp conjugated goat anti rabbit igg
Manufactured by Cell Signaling Technology
Sourced in United States, China
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG is a secondary antibody used in immunoassays and western blotting to detect the presence of rabbit primary antibodies. The HRP enzyme provides a means of signal amplification for sensitive detection.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg, by Cell Signaling Technology (7 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Caspase 9, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Proteasome inhibitor, by Beyotime (3 mentions)
Enhanced chemiluminescent detection reagent, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Western blot chemiluminescent horseradish peroxidase substrate, by Merck Group (3 mentions)
Xrs chemiluminescence detection system, by Bio-Rad (3 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Horse anti mouse igg, by Cell Signaling Technology (2 mentions)
Hrp conjugated horse anti mouse igg, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Mouse monoclonal anti α tubulin antibody, by Merck Group (2 mentions)
Mouse anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Rabbit polyclonal antibody against bcl xl, by Santa Cruz Biotechnology (2 mentions)
Rabbit polyclonal antibody against phospho mcl 1 s159, by Abcam (2 mentions)
Rabbit polyclonal antibody against phospho atm s1981, by Abcam (2 mentions)
39 protocols using horseradish peroxidase hrp conjugated goat anti rabbit igg
1
Subcellular Fractionation and Immunoblotting of HeLa Cells
2
Antibody-based Protein Expression Analysis
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In this study, the antibodies respectively against p38, p-p38, ERK, p-ERK, Akt1/2/3, p-Akt1/2/3, OGT, OGA, CD68 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal Anti-β-O-Linked N-Acetylglucosamine Clone CTD110.6 was obtained from Sigma (St. Louis, MO, USA). Other antibodies including anti-JNK, anti-p-JNK, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).
Chemicals such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris, Tween 20, trifluoroacetic acid, LPS (from Escherichia coli 055:B5), 6-diazo-5-oxo-L-norleucine (DON) and bovine serum albumin were obtained from Sigma. The ABC kit and Agarose wheat germ agglutinin (WGA) were purchased from Vector Laboratories (Lowellville, OH, USA). Thiamet G (Thi G) was obtained from Selleck Chemicals (Houston, TX, USA). RPMI 1640 was purchased from Corning Incorporated (Corning, NY, USA), penicillin, streptomycin and heat-inactivated fetal bovine serum (FBS) was from Gibco (Grand Island, NY, USA). All the other chemicals including sodium dodecyl sulfonate, ammonium persulfate, isopropanol, hydrochloric acid, glycine, sodium chloride and ammonia were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Chemicals such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris, Tween 20, trifluoroacetic acid, LPS (from Escherichia coli 055:B5), 6-diazo-5-oxo-L-norleucine (DON) and bovine serum albumin were obtained from Sigma. The ABC kit and Agarose wheat germ agglutinin (WGA) were purchased from Vector Laboratories (Lowellville, OH, USA). Thiamet G (Thi G) was obtained from Selleck Chemicals (Houston, TX, USA). RPMI 1640 was purchased from Corning Incorporated (Corning, NY, USA), penicillin, streptomycin and heat-inactivated fetal bovine serum (FBS) was from Gibco (Grand Island, NY, USA). All the other chemicals including sodium dodecyl sulfonate, ammonium persulfate, isopropanol, hydrochloric acid, glycine, sodium chloride and ammonia were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
3
Quantitative Western Blot Analysis
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Extracted nuclear protein was denatured in SDS loading buffer and separated by SDS-PAGE (5%-12% acrylamide gradient gels), then transferred onto Amersham Hybond 0.2 PVDF blotting membrane (GE Healthcare Life Sciences, USA). Rabbit anti-NF-κBp65 monoclonal antibody or rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology, Massachusetts, USA) was applied to the membrane for 1 h at 37°C after blocking with 5% nonfat milk. This was followed by incubation with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Cell Signaling Technology, Massachusetts, USA). Finally, the proteins were visualized by a chemiluminescence ECL western blotting analysis system (GE Healthcare, Piscataway, NJ, USA). The protein levels were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and were normalized to β-actin.
4
Investigating Inflammatory Signaling Pathways
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LPS (Escherichia coli, O127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mas receptor antagonist A779 was obtained from AbBiotech (San Diego, CA, USA). ACE2 inhibitor MLN-4760 was a product from EMD Millipore (Darmstadt, Germany). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti-ACE2, anti-p50, anti-p65, and mouse anti-phospho-p50, anti-phospho-p65 and anti-IκBα antibodies were procured from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-/JNK, anti-phospho-JNK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-α and IL-1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang-(1-7) enzyme-linked immunosorbent assay (ELISA) kits were from Kamiya Biomedical (Seattle, WA, USA).
5
Flow Cytometry and Western Blot Analysis
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Anti-CD14-phycoerythrin (PE; MY4; cat. no. CO6603262; Beckman Coulter, Inc.), anti-TLR4-PE (HTA125; cat. no. 12-9917-42; eBioscience; Thermo Fisher Scientific, Inc.), anti-FPR2-PE (GM1D6; cat. no. sc-57141; Santa Cruz Biotechnology, Inc.) and isotype control immunoglobulin G (IgG)-PE (eBM2a; cat. no. 12-4724-42; eBioscience; Thermo Fisher Scientific, Inc.) were used for flow cytometry. Polyclonal anti-P2X7 (cat. no. APR-008; Alomone Labs), normal rabbit IgG (cat. no. PM035; Medical and Biological Laboratories, Ltd.) and PE-conjugated donkey anti-rabbit IgG (cat. no. 406421; BioLegend, Inc.) were also used for flow cytometry. Anti-ICAM-1 (H-108; cat. no. sc-7891; Santa Cruz Biotechnology, Inc.), anti-phosphorylated (p-) NF-κB p65 Ser 536 (93H1; cat. no. 3033; Cell Signaling Technology, Inc.), anti-NF-κB p65 (D14E12; cat. no. 8242; Cell Signaling Technology, Inc.), anti-cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as p21 Waf1/Cip1; 12D1; cat. no. 2947; Cell Signaling Technology, Inc.), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. 12-348; Chemicon International; Merck KGaA) and anti-GAPDH-HRP (5A12; cat. no. 015-25473; Wako Pure Chemical Industries, Ltd.) were used for western blot analysis.
6
Propofol Modulates DENV-Induced Responses
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Propofol (2,6-diisopropylphenol) was purchased from Sigma-Aldrich (St. Louis, MO, USA). An antibody against DENV NS1 (Cat# GTX124280) was purchased from GeneTex (San Antonio, TX); antibodies against phospho-STAT1Tyr701 (Cat# 9167; clone 58D6), STAT1 (Cat# 9172), horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (Cat# 7074S), and HRP-conjugated horse anti-mouse IgG (Cat# 7076S) were purchased from Cell Signaling Technology (Beverly, MA); Alexa Flour 488-conjugated goat anti-mouse antibody (Cat# A-11029) and Hoechst 33258 (Cat# H3569) were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA); antibody against dsRNA (Cat# 10010200) was purchased from SCICONS; antibody against mouse β-actin (Cat# A5441), 4,6-diamidino-2-phenylindole (DAPI; Cat# D9542), the V-ATPase inhibitor bafilomycin A1 (Baf A1; Cat# 19-148), and acridine orange hemi (zinc chloride) salt (Cat# A6014) were purchased from Sigma-Aldrich (St. Louis, MO). According to the manufacturer's instructions, cell cytotoxicity was assessed using Cytotoxicity Detection Kit assays (Roche Diagnostics, Lewes, UK).
7
Western Blot Analysis of Osteogenic Markers
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Cells were lysed in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1500, Cell Signaling Technology), HSPA1A (1:1000; Cell Signaling Technology), Osteocalcin (OCN) (1:1000; abcam, Shanghai, China), RUNX2 (1:1600), COL1A1 (1:1000), or β-catenin (1:1000). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1500; Cell Signaling Technology) was applied as a secondary antibody for 2 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore, Shanghai, China). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).
8
Western Blotting of Brain Tissue Proteins
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Western blotting was performed as previously reported (16 (link)). Brain tissue was lysed using the radioimmunoprecipitation lysis buffer kit (Xi'an Hat Biotechnology Co., Ltd., Xi'an, China). Total protein was determined using a bicinchoninic assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal quantities (25 µg) of protein were resolved by 10% SDS-PAGE with electrophoresis apparatus (DYCZ-40B; Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) and further transferred onto polyvinylidene difluoride membranes, which were soaked with methanol for 30 sec. After blocking with 5% nonfat milk for 1 h at room temperature, samples were probed with MCP-1 (1:1,000; cat. no. 2027S; Cell Signaling Technology Inc., Danvers, MA, USA), CRP (1:1,000; cat. no. 14316S; Cell Signaling Technology Inc.) and GAPDH antibodies (1:1,000; cat. no. 5174T; each, Cell Signaling Technology Inc.) at 4°C overnight, the membrane was washed three times with tris buffered saline tween (TBST; 1×TBS with 0.1% Tween-20) and incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:5,000; cat. no. ab205718, Abcam, Cambridge, UK) at room temperature for 1 h. Finally, the protein bands were visualized using the Immobilon Western Chemiluminescent HRP Substrate kit (EMD Millipore, Billerica, MA, USA) and quantified by an image analysis system (Q550CW; Leica Microsystems GmbH).
9
Antibody Validation for Autophagy Assay
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The anti-SVA rabbit monoclonal antibody (Sangon Biotech, Shanghai, China), anti-SQSTM1/p62 rabbit monoclonal antibody (Beyotime, Shanghai, China), anti-LC3A/MAP1LC3A rabbit monoclonal antibody (Beyotime, Shanghai, China), anti-β-actin mouse monoclonal antibody (Cell Signaling, Danvers, MA, USA), anti-LC3B rabbit polyclonal antibody (Beyotime, Shanghai, China), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), and HRP-conjugated goat anti-mouse IgG (Cell Signaling, Danvers, MA, USA) were used in this study.
10
Immunoblotting and Flow Cytometry Analysis
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The following primary antibodies were used in immunoblotting and flow cytometry: rabbit anti-GAPDH (Santacruz), rabbit anti-phospho-p70 S6 kinase (S6K) (Thr 389) (Cell Signaling), rabbit anti-p70 S6K (Cell Signaling), and APC-conjugated anti-human CD69 (Biolegend). Anti-human CD150 antibody clone 5C6 was generated in our laboratory (Erlenhoefer et al., 2001 (link)). The following labeled secondary antibodies were used: Alexa488-conjugated anti-mouse (Life Technology), Alexa594-conjugated anti-rabbit (Life Technology), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling). For flow cytometry 2 × 105 cells per sample were stained with respective antibodies in FACS buffer (PBS containing 0.4% bovine serum albumin (BSA) and 0.02% sodium azide). Dead cells were stained with PI (Biolegend). Cells were acquired immediately using a LSR II flow cytometer (BD) and the data were analyzed using FlowJo (Cytek Development) software.
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