Protein a peroxidase

Manufactured by Merck Group

Sourced in Germany, United States

Protein A peroxidase is a laboratory reagent used in various immunoassay techniques. It consists of the bacterial protein A conjugated with a peroxidase enzyme. Protein A has a high affinity for the Fc region of immunoglobulin molecules, allowing it to be used as a detection agent in experiments involving antibodies.

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Lab products found in correlation

Immobilon western kit, by Merck Group (2 mentions) Hybond, by Cytiva (2 mentions) Las 4000, by Cytiva (1 mentions) Nitrocellulose membrane, by Merck Group (1 mentions) Odyssey fc, by LI COR (1 mentions) Image studio lite version 5, by LI COR (1 mentions) Immobilon psq, by Merck Group (1 mentions) Irdye 800cw, by LI COR (1 mentions) Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions) Protran, by Cytiva (1 mentions) Hybond c nitrocellulose membrane, by GE Healthcare (1 mentions) Hybond ecl membrane, by GE Healthcare (1 mentions) Beta 3 tubulin antibody, by Abcam (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions) Anti α tubulin t6199, by Merck Group (1 mentions)

Spelling variants of this product

Protein A-horseradish peroxidase conjugate (2 citations) Horseradish peroxidase-linked protein A (2 citations) Peroxidase-conjugated Protein A (2 citations)

7 protocols using protein a peroxidase

Purification of Anti-GST-HCCS Antibodies

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A GST tagged version of the WT human HCCS was expressed, purified,
and injected into rabbits for the production of antisera against GST-HCCS
by Cocalico Biologicals. To purify the antibodies, sodium dextran
sulfate was added to the serum at 0.25% (wt/vol) followed by the addition
of CaCl₂ at 11% (wt/vol) and centrifugation at 11000g. A saturated 76% ammonium sulfate-PBS solution (wt/vol)
was added to the supernatant and incubated with gentle agitation at
4 °C overnight. The antibodies were pelleted by centrifugation
at 11000g, washed in the ammonium sulfate-PBS solution,
resuspended in PBS at 1/10 the starting serum volume, and dialyzed
with PBS overnight. A working solution of the prepared antisera against
GST-HCCS was made and used at a dilution of 1:5000. Protein A peroxidase
(Sigma) was used as a secondary label for detection. The chemiluminescent
signal for anti-GST-HCCS immunoblots was developed using the Immobilon
Western kit (Millipore) and detected by the ImageQuant LAS 4000 mini
detection system (Fujifilm-GE Healthcare).

Babbitt S.E., San Francisco B., Bretsnyder E.C, & Kranz R.G. (2014). Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme. Biochemistry, 53(32), 5261-5271.

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Western Blot Protein Transfer and Detection

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The antigens were transferred from SDS-PAGE gel to nitrocellulose membrane (Millipore, Germany) at 45 mA for 45 min. The nonspecific binding sites were blocked by membrane incubation in a 5% BSA (bovine serum albumin) solution (Merck Millipore, Germany) for 30 min. The membrane was washed 3 times with phosphate buffered saline pH 7.2 (Merck Millipore, Germany). Purified antibody in a 1:100 dilution was added to the membrane and incubated for 2 h. The membrane was washed for 2 h with phosphate buffer and incubated with protein A peroxidase (Sigma Aldrich, Germany) as secondary antibody in a 1:1000 dilution. The nitrocellulose membranes were exposed to 15 mL of substrate solution (Thermo Fisher Inc., Germany) for 5 to 15 min. Test was repeated using commercial antibody (Roche, Switzerland).

Maboudi K., Hosseini S.M., Sepahi M., Yaghoubi H, & Hadadian S. (2017). Production of Erythropoietin-Specific Polyclonal Antibodies. Iranian Journal of Biotechnology, 15(1), 50-57.

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Heme Abundance Quantification by Immunoblotting

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Heme stains were performed as previously described by [54 (link)] with the following modifications. Proteins were transferred to 0.2 μm Immobilon P^SQ (Millipore). Two membranes were layered to capture free heme that transferred through the first membrane. Imaging was performed with a LAS-1000 Plus (Fujifilm-GE Healthcare) or LI-COR Odyssey Fc (LI-COR Biosciences). Heme abundance was determined by densitometry performed with ImageJ [55 ] or Image Studio Lite Version 5.2 software (LI-COR Biosciences). Note that the total free heme value was determined by analysis of heme on both membranes. For immunoblots, proteins were separated via SDS-PAGE, transferred to 0.45 μm Amersham Protran nitrocellulose (GE Healthcare Life Sciences) and probed with the following antibodies: CcmE – α-CcmE at 1:15000 [24 (link)], GST:CcmC – α-GST at 1:5000 (Sigma-Aldrich). Protein A peroxidase (Sigma-Aldrich) and developed with Immobilon Western Chemiluminescent HRP substrate (Millipore) or IRDye 800 CW (LI-COR Biosciences) were used as secondary labels. Imaging and quantitation was performed as above and total proteins stains were performed with Coomassie Blue or SYPRO Ruby Protein. Note that for ease of viewing, Coomassie stains are false colored blue, heme stains false colored red and western blots false colored green.

Sutherland M.C., Jarodsky J.M., Ovchinnikov S., Baker D, & Kranz R.G. (2018). Structurally Mapping Endogenous Heme in the CcmCDE Membrane Complex for Cytochrome c Biogenesis. Journal of molecular biology, 430(8), 1065-1080.

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Cytochrome c Purification and Analysis

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Heme stains and immunoblots were performed as described previously (Feissner et al., 2003 (link); Feissner et al., 2006b (link)). Proteins were separated by 12.5 % SDS-PAGE and transferred to Hybond C nitrocellulose membranes (GE Healthcare). For immunoblots, anti-CcmF antibodies were used at a dilution of 1:10000, anti-CcmE antibodies at 1:10000, anti-cytochrome c4 antibodies at 1:10000, and anti-CcmH antibodies at 1:5000. Protein A peroxidase (Sigma-Aldrich) was used as the secondary label. The chemiluminescent signal for heme stains was developed using the SuperSignal Femto kit (Thermo Scientific) or, for immunoblots, the Immobilon Western kit (Millipore), and detected with an LAS-1000 Plus detection system (Fujifilm-GE Healthcare). The abundance of holocytochrome c4 was determined by pyridine extraction as described in (Berry and Trumpower, 1987 (link)), or by densitometry analysis of the chemiluminescent signal from heme staining using the Science Lab 99-Image Gauge version 3.4 software (Fujifilm-GE Healthcare) as described in (Richard-Fogal et al., 2007 (link)). Protein purity was assessed by Coomassie Blue staining of SDS-PAGE.

Francisco B.S., Sutherland M.C, & Kranz R.G. (2014). The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE. Molecular microbiology, 91(5), 996-1008.

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ELISA for Canine Leishmaniasis Diagnosis

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All samples were tested for the presence of IgG against L. infantum through an in-house enzyme-linked immunosorbent assay (ELISA), using a technique described previously [44 (link), 45 (link)]. Again, serial samples from a single dog were tested in parallel on the same plate. Briefly, dog plasma samples diluted at 1:400 were incubated in titration plates (Costar® Corning®, New York, USA) previously coated with sonicated whole promastigotes at a protein concentration of 20 μg/ml in 0.05 M carbonate buffer at pH 9.6. Protein A peroxidase (1:30,000, Sigma-Aldrich®, St. Louis, Missouri, USA) was used as conjugate and reactions were stopped with H₂SO₄ 3M when a pre-determined calibrator control serum reached an optical density of 450 at 450 nm. Sample optical densities were read at 492 nm. All samples were run in duplicate and the calibrator, positive and negative sera were included in all plates. Results were expressed in standard units (U) compared to a calibrator control sample set arbitrarily at 100U. The positivity cut-off was established at 24U.

Velez R., Spitzova T., Domenech E., Willen L., Cairó J., Volf P, & Gállego M. (2018). Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum. Parasites & Vectors, 11, 545.

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Western Blot and Immunohistochemistry Analysis

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Whole-cell protein extracts were prepared from cells lysed in RIPA buffer. Samples were separated through SDS PAGE gels, transferred to Hybond ECL membranes (GE Healthcare), and treated with appropriate antibodies. Staining was performed by the SuperSignal Chemiluminescent Substrate from Pierce. ImageJ software (http://rsbweb.nih.gov/ij/) was used for densitometric analysis; western blot quantification was normalized against loading controls. Immunohistochemical detection was performed as described (Annibali et al. 2012 (link)).
Antibodies used: phospho-Akt(Ser473) and phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibodies were from Cell Signaling, Vgf antibody [24] was kindly provided by A. Levi, monoclonal anti-β-Actin–peroxidase clone Ac-15 (A3854) and anti-α-Tubulin (T6199) were from Sigma, beta III tubulin antibody was from Abcam, and mouse monoclonal anti-App (22C11) from Millipore. Horseradish peroxidase secondary antibodies were from Chemicon and Protein A peroxidase from Sigma.

Montalban E., Mattugini N., Ciarapica R., Provenzano C., Savino M., Scagnoli F., Prosperini G., Carissimi C., Fulci V., Matrone C., Calissano P, & Nasi S. (2014). MiR-21 is an Ngf-Modulated MicroRNA That Supports Ngf Signaling and Regulates Neuronal Degeneration in PC12 Cells. Neuromolecular Medicine, 16(2), 415-430.

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Crude L. infantum ELISA for Seroconversion

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A crude total L. infantum antigen in house enzyme linked immunosorbent assay (CTLA-ELISA) was used to detect IgG antibodies to L. infantum in trial dogs. The technique used has been previously described (Riera et al., 1999; (link)Velez et al., 2019) (link). Briefly, dog plasma samples diluted at 1:400 were incubated in titration plates (Costar®) previously coated with sonicated whole promastigotes at a protein concentration of 20 µg/mL in 0.05 M carbonate buffer at pH 9.6.
Protein A peroxidase (1:30,000, Sigma®) was used as conjugate and reactions were stopped with H2SO4 3M. Results were expressed in standard units (U) compared to a calibrator control sample set arbitrarily at 100U. The cut-off was established at 24U (mean + 4 standard deviations of U of sera of dogs from non-endemic areas).
Serological assessments were performed at eight time points throughout the study: before each vaccine dose (T0, Vac2, Vac3) and at one (1M), four (4M), six (6M), nine (9M) and 12 months (12M) after vaccination completion. An increase of four-fold ELISA units when compared with the same dog's basal values (ELISA units measured at T0) was considered evidence of seroconversion to L. infantum (Solano-Gallego et al., 2009) (link).

Velez R., Domenech E., Rodríguez-Cortés A., Barrios D., Tebar S., Fernández-Arévalo A., Aguilar R., Dobaño C., Alberola J., Cairó J, & Gállego M. (2020). Evaluation of canine leishmaniosis vaccine CaniLeish® under field conditions in native dog populations from an endemic Mediterranean area-A randomized controlled trial. Acta tropica, 205.

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