Peroxidase conjugated secondary antibody

Manufactured by Promega

Sourced in United States

The Peroxidase-conjugated secondary antibody is a laboratory reagent used for the detection and visualization of target proteins in various immunoassays. It consists of a secondary antibody that is chemically linked to the enzyme peroxidase. This enzyme catalyzes a reaction that produces a colored or chemiluminescent signal, enabling the identification and quantification of the target protein in the sample.

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Lab products found in correlation

Protease inhibitor cocktail, by Santa Cruz Biotechnology (3 mentions) Fluorchem sp digital imager, by Bio-Techne (3 mentions) Ecl kit, by Cytiva (2 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions) Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions) Uvp biospectrum 500 imaging system, by Analytik Jena (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Image lab, by Bio-Rad (1 mentions) Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) The antibody against gapdh, by Santa Cruz Biotechnology (1 mentions) Phospho plc γ1 tyr783, by Cell Signaling Technology (1 mentions) Mouse anti penta his, by Qiagen (1 mentions) Protease inhibitor cocktail, by Abcam (1 mentions) Protein extraction kit, by Qiagen (1 mentions) Polyvinylidene fluoridehydrophobic membrane, by Merck Group (1 mentions) Skimmed milk, by Solarbio (1 mentions) Ecl detection system, by Cytiva (1 mentions) Sc 7210, by Santa Cruz Biotechnology (1 mentions) Sc 8979, by Santa Cruz Biotechnology (1 mentions)

11 protocols using peroxidase conjugated secondary antibody

Western Blot Protein Analysis Protocol

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Protein (10–20 μg/lane) was separated by 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BioRad) and transferred to polyvinylidene difluoride membranes (Bio-Rad) by electroblotting. Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and then incubated with primary antibodies (Supplementary Table 5) at 4 °C overnight. Membranes were then washed in TBST (20 mM Tris pH7.5, 150 mM NaCl, 0.1% Tween20) and incubated with appropriate peroxidase-conjugated secondary antibodies (Promega and Cell Signaling Technology) for 1 h at room temperature. Signals were visualized with an ECL Kit (Amersham Bioscience or BioRad). Band intensities were quantified using Image Lab (Bio-Rad).

Ning J.F., Stanciu M., Humphrey M.R., Gorham J., Wakimoto H., Nishihara R., Lees J., Zou L., Martuza R.L., Wakimoto H, & Rabkin S.D. (2019). Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma. Nature Communications, 10, 2910.

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BN-PAGE Analysis of Mitochondrial Complexes

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For BN-PAGE analysis50 (link), mitochondrial extracts (starting from 40–100 μg mitochondrial proteins) solubilized with digitonin (digitonin to protein ratio of 10 g/g) were separated on NativePAGE 3–12% Bis-Tris gels (Invitrogen, USA) or on homemade blue native gradient gels (5–10%, 4–16.5% or 6–16.5% polyacrylamide). After electrophoresis, gels were either stained with Coomassie blue, incubated in a solution of 5 mM ATP, 5 mM MgCl₂, 0.05% lead acetate, 50 mM glycine–NaOH, pH 8.4 to detect ATPase activity51 (link), or transferred to nitrocellulose or PVDF membranes and analysed by western blotting52 (link). Membranes were first incubated with peptide antibodies against Cox4, Atp4 or F₁β, or polyclonal antibodies raised against cytochrome b. Subsequently, they were incubated with peroxidase-conjugated secondary antibodies at a 1:5,000 dilution (Promega or Sigma) and revealed using enhanced chemiluminescence.

Aiyar R.S., Bohnert M., Duvezin-Caubet S., Voisset C., Gagneur J., Fritsch E.S., Couplan E., von der Malsburg K., Funaya C., Soubigou F., Courtin F., Suresh S., Kucharczyk R., Evrard J., Antony C., St.Onge R.P., Blondel M., di Rago J.P., van der Laan M, & Steinmetz L.M. (2014). Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders. Nature Communications, 5, 5585.

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Western Blot Analysis of PAK-1 Levels

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Cell lysates from different cell lines were collected in RIPA buffer, which contained a protease inhibitor cocktail (Santa Cruz Biotechnology, Inc). The BCA assay was used to determine protein concentrations. Samples of 40 µg of protein were separated using SDS‐PAGE and then transferred to nitrocellulose membranes that were then blocked in 5% (w/v) nonfat dry milk in Tris‐buffered saline‐Tween 20 (TBST). After 2 hours of blocking, the membranes were incubated with a rabbit PAK‐1 antibody (Cell Signaling Technology) at a dilution of 1:1000 in 1% (w/v) BSA TBST overnight. The antibody against GAPDH (Santa Cruz Biotechnology Inc) was used at a dilution of 1:4000 in 1% (w/v) BSA in TBST for 1 hour. Membranes were then incubated with the appropriate peroxidase‐conjugated secondary antibodies (Promega,) used at a dilution of 1:2500. The membranes were then washed with TBST three times for 10 minutes each. Bands were visualized using chemiluminescent substrates (Thermo Scientific) and imaged with a FluorChem SP digital Imager (Alpha Innotech). Immunoblot analysis was performed on protein samples from at least three different passages (n = 3) of control and KD cells. Densitometry was performed using National Institutes of Health Image J software.

Najahi‐Missaoui W., Quach N.D., Jenkins A., Dabke I., Somanath P.R, & Cummings B.S. (2019). Effect of P21‐activated kinase 1 (PAK‐1) inhibition on cancer cell growth, migration, and invasion. Pharmacology Research & Perspectives, 7(5), e00518.

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Western Blot Protein Analysis

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Protein (10–15 μg/lane) was separated by 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BioRad) and transferred to polyvinylidene difluoride membranes (Bio-Rad) by electro blotting. Membranes were blocked with 5% nonfat skim milk for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight. Membranes were then washed in TBST (20 mM Tris pH7.5, 150 mM NaCl, 0.1% Tween20) and incubated with appropriate peroxidase-conjugated secondary antibodies (Promega and Cell Signaling Technology) for 1 h at room temperature. Signals were visualized with an ECL Kit (Amersham Bioscience or BioRad).

Li M., Kirtane A.R., Kiyokawa J., Nagashima H., Lopes A., Tirmizi Z.A., Lee C.K., Traverso G., Cahill D.P, & Wakimoto H. (2020). Local targeting of NAD+ salvage pathway alters the immune tumor microenvironment and enhances checkpoint immunotherapy in glioblastoma. Cancer research, 80(22), 5024-5034.

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Antibody Immunostaining Protocol

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Mouse monoclonal antibodies to GM130 and Golgin97 were from Transduction Laboratories and Molecular Probes, respectively. Sheep anti-TGN46 was purchased from Serotech and mouse anti-PentaHis was from Qiagen. Antibodies against PLCγ1 and phospho-PLCγ1 (Tyr783) were purchased from Cell Signalling. Rabbit polyclonal anti-GFP, anti-FLAG and anti-ectodomain of VSV-G were purchased from Sigma-Aldrich. Cyanine 3-conjugated mouse and rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories and Alexa Fluor 488, 546 and 647-conjugated anti-mouse, anti-rabbit and anti-sheep antibodies were obtained from Molecular Probes. Peroxidase-conjugated secondary antibodies were from Promega.

Sicart A., Katan M., Egea G, & Sarri E. (2015). PLCγ1 participates in protein transport and diacylglycerol production triggered by cargo arrival at the Golgi. Traffic (Copenhagen, Denmark), 16(3).

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Western Blot Analysis of Total Protein

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Total protein extracts were obtained by cell lysis in LB-SDS buffer (25% Tris HCl 0.5 M pH 6.8, 25% SDS 10%, 50% ddH20). Protein concentrations were quantified by BCA Protein Assay kit (Thermofisher) and equal amounts of total cellular lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were blocked by incubation in 10% BSA. The membranes were incubated in the specific properly diluted primary antibody and then in the appropriate peroxidase-conjugated secondary antibody (Promega). Signal detection was done by enhanced chemioluminescence (ECL, Amersham Biosciences, GE Healthcare, Chicago, IL, USA). Band densitometry analysis was done with ImageJ software (NIH, Bethesda, MD, USA). All whole western blots are available in Figure S6.

Rizzolio S., Corso S., Giordano S, & Tamagnone L. (2020). Autocrine Signaling of NRP1 Ligand Galectin-1 Elicits Resistance to BRAF-Targeted Therapy in Melanoma Cells. Cancers, 12(8), 2218.

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Protein Expression Analysis Protocol

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Cellular protein samples were isolated with a Protein Extraction Kit (Qiagen, GmbH, Hilden, Germany) according to the kit's manual and supplemented with a protease inhibitor cocktail (Abcam, Cambridge, UK). Protein samples were separated with 10% SDS-PAGE gel and were transferred to a polyvinylidene fluoride hydrophobic membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skimmed milk (Solarbio, Beijing, China) overnight at 4°C to cover the nonspecific binding sites. Then the membrane was inoculated with the rabbit anti-mouse KLF-4 (BM0485, Abzoom Biolabs, Dallas, TX, USA; 1 : 300), PAI-1 (sc-8979, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), E-cadherin (sc-7870, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 500), Collagen I (ab21286, Abcam, Cambridge, UK; 1 : 200), mouse anti-mouse α-Smooth Muscle Actin (α-SMA) (sc-53142, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 200), or rabbit anti-mouse β-actin (sc-7210, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 800) at room temperature for 2 hours. The specific binding to each protein marker was presented with the incubation with the peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA) and the electrochemiluminescence (ECL) detection system (Amersham, Uppsala, Sweden). The protein level was presented as a ratio to β-actin.

Sun F, & Hu K. (2015). Krüppel-Like Factor 4 Inhibits the Transforming Growth Factor-β1-Promoted Epithelial-to-Mesenchymal Transition via Downregulating Plasminogen Activator Inhibitor-1 in Lung Epithelial Cells. Disease Markers, 2015, 473742.

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Western Blot Analysis of PAK-1 Protein

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Proteins from different cell lines were collected in RIPA buffer, which contained protease inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein concentrations were determined using the BCA assay. Protein samples of 40 μg were separated on SDS-PAGE gels and transferred to nitrocellulose membranes, which were then blocked in 5% (w/v) nonfat dry milk in Tris-buffered saline–Tween 20 (TBS-T) for 2 hrs. The membranes were then incubated with a rabbit PAK-1 antibody at a dilution of 1:500 in 1% (w/v) BSA TBS-T overnight. Antibodies against GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used at a dilution of 1:200 in 1% (w/v) BSA in TBST for 1 hr. Membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody (Promega, Madison, WI) used at a dilution of 1:2500. The membrane was then washed with TBS-T three times for 10 minutes each and imaged with a Fluorchem SP digital imager (Alpha Innotech, San Leandro, CA, USA). Densitometry was performed using National Institutes of Health Image J software.

Al-Azayzih A., Missaoui W.N., Cummings B.S, & Somanath P.R. (2016). Liposomal-mediated delivery of the p21 activated kinase-1 (PAK-1) inhibitorIPA-3limits prostate tumor growth in vivo. Nanomedicine : nanotechnology, biology, and medicine, 12(5), 1231-1239.

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Immunoblot Analysis of Protein Levels

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Immunoblot analysis and protein determination experiments were performed as previously described(58 (link)). Briefly, monolayers of cells were washed in phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonylfluoride and 1 mM sodium orthovanadate and then lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA Assay (Pierce). Forty micrograms of total protein was loaded per lane and separated by SDS-PAGE. After transfer, the nitrocellulose membrane (Invitrogen) was blocked with either 5% nonfat-milk or 5% BSA in TBST before addition of primary antibodies and followed with peroxidase-conjugated secondary antibody (Promega). Protein bands were detected using SuperSignal Chemiluminescent Substrate (Pierce) with a UVP BioSpectrum 500 Imaging System.

Roos A., Dhruv H.D., Peng S., Inge L.J., Tuncali S., Pineda M., Millard N., Mayo Z., Eschbacher J.M., Loftus J.C., Winkles J.A, & Tran N.L. (2018). EGFRvIII-Stat5 Signaling Enhances Glioblastoma Cell Migration and Survival. Molecular cancer research : MCR, 16(7), 1185-1195.

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Immunoblot Analysis of Protein Expression

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Immunoblot analysis and protein determination experiments were performed as previously described [21 (link)]. Briefly, monolayers of cells were washed in phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonylfluoride and 1 mM sodium orthovanadate and then lysed in 2× SDS sample buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA Assay (Pierce). Thirty micrograms of total protein was loaded per lane and separated by SDS-PAGE. After transfer, the nitrocellulose membrane (Invitrogen) was blocked with either 5% nonfat-milk or 5% BSA in TBST before addition of primary antibodies and followed with peroxidase-conjugated secondary antibody (Promega). Protein bands were detected using SuperSignal Chemiluminescent Substrate (Pierce) with a UVP BioSpectrum 500 Imaging System.

Dhruv H.D., Roos A., Tomboc P.J., Tuncali S., Chavez A., Matthews I., Berens M.E., Loftus J.C, & Tran N.L. (2015). Propentofylline inhibits glioblastoma cell invasion and survival by targeting the TROY signaling pathway. Journal of neuro-oncology, 126(3), 397-404.

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