MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide), protease inhibitor cocktail, an LC-3B primary antibody, metformin hydrochloride, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against TIMM23, and NDUFS3 as well as a JC-10 Mitochondrial Membrane Potential Assay Kit were purchased from Abcam (Cambridge, UK). Secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Cell Signaling Technology (Danvers, MA, USA). Bradford Protein Assay Reagent was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Enhanced Chemiluminescence (ECL) Western blotting Substrate was purchased from Pierce Biotechnology (Rockford, IL, USA). An RNA extraction kit, High Capacity Reverse Transcription Kit, and Kapa SYBR® FAST qPCR Kit (ABI Prism) were purchased from Qiagen (Germantown, MD, USA), Applied BioSystems (Foster City, CA, USA) and Kapa Biosystems (Sigma-Aldrich, St. Louis, MO, USA), respectively.
Secondary antibodies conjugated to horseradish peroxidase hrp
Manufactured by Cell Signaling Technology
Sourced in United States
Secondary antibodies conjugated to horseradish peroxidase (HRP) are laboratory reagents used in various immunoassays and detection methods. These antibodies bind to primary antibodies and the attached HRP enzyme can catalyze a chromogenic or chemiluminescent reaction, enabling the detection and visualization of target proteins or biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay reagent, by Bio-Rad (1 mentions)
Enhanced chemiluminescence ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Jc 10 mitochondrial membrane potential assay kit, by Abcam (1 mentions)
Metformin hydrochloride, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Lc 3b primary antibody, by Merck Group (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Epr1942, by Abcam (1 mentions)
Syto13, by Thermo Fisher Scientific (1 mentions)
Anti β actin a2228, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Deadend fluorometric terminal deoxynucleotidyl transferase dutp nick end labeling tunel system, by Promega (1 mentions)
Anti p drp 1 ser616, by Cell Signaling Technology (1 mentions)
Anti parkin ab15954, by Abcam (1 mentions)
Anti tfeb antibody a303 673a, by Fortis Life Sciences (1 mentions)
Anti erk1 2, by Merck Group (1 mentions)
Phospho stat3 y705, by Cell Signaling Technology (1 mentions)
6 protocols using secondary antibodies conjugated to horseradish peroxidase hrp
1
Mitochondrial Function Evaluation Protocol
2
Western Blot Quantification Protocol
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Protein concentration in cellular extracts was determined by the standard Bradford procedure (Bradford 1976 (link)). Samples of identical protein amount (30 µg) were separated by 10 % polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) according to Laemmli (1970 ), as a molecular mass marker served PageRuler™ Prestained Protein Ladder (Fermentas). This was followed by transfer to nitrocellulose membrane, using the procedure described elsewhere (Towbin et al. 1979 (link)). Monoclonal rabbit anti-gelsolin antibodies (Abcam, clone EPR1942) at dilution 1:5,000 were used to visualize gelsolin band on nitrocellulose. β actin and β tubulin recognized by monoclonal mouse anti-β actin (Sigma, clone AC-15) and anti-β tubulin (TUB 2.1) antibodies, were used as reference proteins. Secondary antibodies conjugated to horseradish peroxidase (HRP) were applied according to the manufacturer’s protocols (Cell Signaling). Immunoblots were developed using the Western blotting Luminol Reagent (Santa Cruz Biotechnology), photos of blots were taken with ChemiDoc™ MP System (Bio-Rad) and further analyzed with the help of ImageLab 4.0 software (Bio-Rad).
3
Mitochondrial Dysfunction and Cell Death Pathway Analysis
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Bafilomycin A1 (B1793), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (C2759), propidium iodide (P4170), oligomycin (O4876), 2-[2-[4(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) (C2920), antimycin A (AA) (A8674), and rotenone (R8875) were purchased from Sigma-Aldrich. SYTO-13 (S7575) was purchased from Thermo Fisher Scientific. The Masson’s Trichrome staining kit was obtained from RAL Diagnostic (RAL-361350-0000). The DeadEndTM Fluorometric Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System was purchased from Promega (G3250). Anti-cleaved-caspase 3 (Asp175) (9664), anti-LC3B (2775), anti-VDAC1 (4661), anti-ATG7 (2631), anti-Beclin 1 (3728), anti-GRP75 (2816), and anti-p-Drp-1 (Ser616) (3455) antibodies were from Cell Signaling Technology. Anti-β-actin (A2228) and anti-SQSTM1/P62 (P0067) were from Sigma-Aldrich. Anti-TOMM 40 antibody (H-300) was from Santa Cruz Biotechnologies. Anti-MOMA 2 antibody (MAB1825) was from Merck Millipore, anti-Parkin (ab15954) and anti-PINK1 (ab74487) were from Abcam. Anti-PGC-1α antibody (NBP1-04676) was from NOVUS Biologicals. Anti-TFEB antibody (A303-673A) was from Bethyl Laboratories. Smooth muscle actin rabbit polyclonal antibody (RB-9010-P) was from Thermo Fisher Scientific. Secondary antibodies conjugated to horseradish peroxidase (HRP) were from Cell Signaling Technology.
4
Procalcitonin Sandwich ELISA Protocol
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A 50 mM carbonate-bicarbonate buffer containing the anti-PCT antibody (1: 500 dilution) (Cell Signaling Technology, Boston, USA) was used to coat a 96-well plate at 4°C for 12 hours, followed by treating the plate for another 60 min using PBS containing 4% bovine serum albumin (BSA). Subsequently, the plate was washed with PBS containing 0.1% Tween-20, and 50 μl of a mixture containing purified PCT antigen and clinical samples were distributed among each of 96 wells. The plate was then incubated for 120 min at 37°C and washed. Then, 100 μl of PBS containing 1% BSA and 1: 1500 dilution of the anti-PCT antibody was added to each well, and the plate was incubated for 60 min at 37°C. The plates were washed with 1% BSA (LI-COR Inc., Lincoln, NE, USA) and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) (1: 3000 dilution) (Cell Signaling Technology, Boston, USA). In the presence of O-phenylenediamine, the HRP activity in each well of the plate was measured at 492 nm absorbance using a microplate reader and H2O2 served as the substrate for the enzyme. Each experiment was performed in triplicate.
5
Antibody Procurement for Heparan Sulfate Research
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Rabbit antibody to HS3ST2 and mouse antibody to HS3ST3A (clone E-12) were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Santa-Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Mouse antibodies to HS3ST3B, HS3ST4 and survivin were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies to phospho-Akt(S473), Akt, phospho-c-Src(Y416), c-Src, phospho-ERK1/2(T202/Y204), phospho-STAT3(Y705), STAT3, I-κB, phospho-NF-κB p65(S536), NF-κB p65, XIAP and secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Cell Signaling Technology (Danves, MA, USA). Mouse antibody to GAPDH was from Santa Cruz. HS4C3 and MBP49 antibodies were provided by T. Van Kuppevelt (University of Nijmegen, Netherlands) and used as described in [23 (link)]. Other chemicals and antibodies, including anti-ERK1/2, were from Sigma-Aldrich (Darmstadt, Germany) unless otherwise specified.
6
Kinase Activation Signaling Pathway
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All reagents for cell culture were obtained from Life Technologies (Grand Island, NY).
Antibodies against phospho-p44/42 MAP kinase (D.13.14.4E) and GAPDH (14C10) were obtained from Cell Signaling Technologies (Danvers, MA). Recombinant platelet factor 4 (PF4)
was purchased from Abcam (Cambridge, MA). Secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Cell Signaling Technologies. The MEK inhibitors PD98059 and PD184352 were purchased from Calbiochem (Billerica, MA) and Axon Medchem (Reston, VA), respectively, and dissolved in a dimethyl sulfoxide (DMSO) vehicle.
Antibodies against phospho-p44/42 MAP kinase (D.13.14.4E) and GAPDH (14C10) were obtained from Cell Signaling Technologies (Danvers, MA). Recombinant platelet factor 4 (PF4)
was purchased from Abcam (Cambridge, MA). Secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Cell Signaling Technologies. The MEK inhibitors PD98059 and PD184352 were purchased from Calbiochem (Billerica, MA) and Axon Medchem (Reston, VA), respectively, and dissolved in a dimethyl sulfoxide (DMSO) vehicle.
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