To measure IgG, IgG1 and IgG2a responses against CP4 in mouse sera and IgA in vaginal washes, purified CP4 membrane proteins resuspended in sodium carbonate-bicarbonate buffer (pH 9.6) were used to coat polystyrene 96-well flat-bottom microtiter plates (Dynatech Laboratories Inc., Chantilly, VA) with 100 ng/well. After incubation at 4°C overnight, the plates were blocked with sea block blocking buffer (Fisher). The serum and vaginal washes were serially diluted and 100 µl of diluted sample was added to duplicate wells and incubated for 1 h at 37°C. After three times PBS-0.05% Tween-20 washing, the plates were incubated with biotinylated goat anti-mouse IgG, IgG1, IgG2a, or IgA (Southern Biotechnology Inc., Birmingham, AL) antibodies diluted 1:10,000 for 1 h at 37°C. Wells were developed with streptavidin horseradish peroxidase conjugate (Invitrogen, Carlsbad, CA) at the ratio of 1:6,000, followed by 2,2--azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Sigma). Color development (absorbance) was recorded at 405 nm using a SpectraMax M2 Multi-Mode Microplate Reader (Molecular Devices, LLC). Titers were recorded as the last dilution that resulted in 0.1 greater than background and expressed as the reciprocal log2 values.
Streptavidin horseradish peroxidase conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin-horseradish peroxidase conjugate is a complex of the protein streptavidin and the enzyme horseradish peroxidase. It is used in various bioanalytical techniques to detect and quantify target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Spectramax m2 multi mode microplate reader, by Molecular Devices (1 mentions)
Sephadex g 25 column, by GE Healthcare (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose column, by GE Healthcare (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Pod substrate, by Roche (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
Tmb elisa substrate, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti flag, by Merck Group (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
16 protocols using streptavidin horseradish peroxidase conjugate
1
Measuring Humoral Immune Responses to CP4 in Mice
2
Monoclonal Antibody Production against Bacillus cereus
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B. cereus ATCC11778 and related species strains analyzed in this study were stored at −80 °C. Incomplete Freund’s adjuvant, 50% polyethylene glycol-1450 (PEG1450), paraformaldehyde, RPMI1640, fetal calf serum (FCS), hypoxanthine/aminopterin/thymidine (HAT), hypoxanthine/thymidine (HT), methyl cellulose, 3, 3, 5, 5-tetramethylbenzidine (TMB), and mouse monoclonal antibody ISO2-1 kits were purchased from Sigma-Aldrich (Shanghai, China). Goat anti-mouse immunoglobulin horseradish peroxidase conjugate, goat anti-rabbit immunoglobulin horseradish peroxidase conjugate (IgG–HRP) were purchased from Univ-bio (Shanghai, china) and streptavidin-horseradish peroxidase conjugate were purchased from Invitrogen (Shanghai, china). The Sephadex G-25 column and protein A-sepharose columns were purchased from General Electric Company (GE) (Shanghai, China). All other reagents were of analytical grade. The ELISA was conducted in EIA 1 × 8 StripwellTM Plates, No Lid, 42592 (Costar, USA) using a 12-channel pipette (50 ~ 300 μL, Thermo Lab Systems Co. Ltd., Shanghai, China). The absorbance at 450 nm was scanned in each well with a Varioskan Flash (Thermo, USA). BABL/c murine myeloma cells SP2/0 were conserved by our laboratory. BABL/c mice were obtained from Charles River Company (Beijing, China). The amounts (cells/mL) of B. cereus were counted in a Petrof Hausser bacteria chamber.
3
Immunohistochemical Analysis of MUC1 in Colon Tissues
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For MUC1 detection, 6 μm thick cryostat sections of normal colon as well as colonic tissue with tubular adenoma or squamous metaplasia were used. Sections were pretreated for 30 min with 0.3% hydrogen peroxide at room temperature to eliminate endogenous peroxidase and washed three times with Dulbecco’s PBS. All endogenous biotin, biotin receptors, or avidin binding sites were blocked using the VECTASTAIN ABC HRP Kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s protocol. The slides were incubated overnight at 4 °C with biotin-conjugated primary Abs or isotype control Abs at 5 μg/mL diluted in 3% BSA in PBS. Subsequently, Streptavidin Horseradish Peroxidase Conjugate (Invitrogen) was used at 1/200 dilution. Sections were counterstained with H&E. Immunoreactivity against MUC1 Ab was assessed under a microscope (Leica Microsystems, Wetzlar, Germany). To measure MUC1-glycoform expression in tumors, semi-quantitative analysis of immunohistochemically stained sections was performed by roughly estimating the percentage of MUC1-positive cells as compared to all epithelial cells.
4
Competitive Binding Assay for Antibody Characterization
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Competitive binding assays were performed as described previously 14 (link). In brief, the NUNC plate was pre-coated with RBD-VLP 40 (link) overnight at 4ºC. After the plate was washed and blocked with 5% dried skimmed milk in PBS for 1 h at room temperature, the mixture of biotinylated antibody (EZ-Link Sulfo-NHS-LC-biotin, Life Technologies, United States) and at least ten-fold excess of competing antibody was added to the plate and incubated for 1 h. After washing, the plate was incubated with Streptavidin-Horseradish Peroxidase conjugate (Life Technologies, United States) for another one hour. After the plate was washed, the signal was developed using the POD substrate (Roche, Switzerland) and the reaction was stopped with 1M H2SO4. The OD450 value was measured using a Clariostar plate reader (BMG Labtech, Germany). The mean and 95% confidence interval of four replicates were calculated. Competition was measured as: (X-minimum binding/(maximum binding-minimum binding), where X is the binding of the biotinylated antibody in the presence of competing antibody. Minimum binding is the self-blocking of the biotinylated antibody or background binding. Maximum binding is binding of biotinylated antibody in the presence of non-competing antibody (anti-influenza neuraminidase antibody Z3B2).
5
Quantification of POMC-derived ACTH Secretion
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Full-length (+3799/+3940 and +6828/+7499), Del-A (+6929/+7499), and Del-B (+6996/+7499) POMC cDNA fragments were PCR amplified and cloned into Hind III and Xho I sites in the pMF neo expression vector. A total of 1 µg of plasmids was transfected into 5 × 105 COLO320 cells by lipofectamine 2000 (Invitrogen) and cultured for 48 hours, and the cultured media was collected for ELISA assays. Ninety-six–well plates were coated with anti-ACTH antibody (1 ng/well, Abcam, catalog No. ab20358) overnight at 4 °Ϲ and then blocked with 1% bovine serum albumin/phosphate-buffered saline for 1 hour at room temperature. Next, 50 µL of cultured supernatant was added into 2 wells and incubated for 1 hour at room temperature. After washing with phosphate-buffered saline, biotin-conjugate monoclonal antibody to hemagglutinin (HA) (1 µg, Sigma-Aldrich, catalog No. B9183) was added and incubated for 1 hour at room temperature. Antibody binding was detected with Streptavidin–horseradish peroxidase conjugate (Life Technologies, catalog No. SNN1004), TMB-ELISA substrate (Thermo Fisher Scientific, catalog No. 34028) and Stop Solution TMB (Thermo Fisher Scientific, catalog No. N600). Absorbance at 450 nm was measured by an ELISA plate reader, and a calibration curve used to calculate ACTH concentrations.
6
Western Blotting and Immunofluorescence Antibody Panel
7
Anti-CD0873 Antibody Detection by ELISA
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Anti-CD0873 IgG and IgA were detected in serum and feces, respectively, by indirect ELISA as described previously (38 (link)). Briefly, 96-well microtiter plates (MaxiSorp, Nunc) were coated with 5 μg of recombinant purified CD0873 protein. Fecal supernatants were tested at a dilution of 1:2 and mouse sera at a dilution of 1:20. After washing, anti-CD0873 antibodies were detected by successive incubations for 30 min at 37 °C with a goat anti-mouse IgG or IgA conjugated to biotin (1:20,000 and 1:10,000 dilution, respectively; Sigma) and for 30 min at 37 °C with a streptavidin-horseradish peroxidase conjugate (1:5,000 dilution; Thermo Scientific). All samples were treated simultaneously and tested in duplicate to avoid inter-assay variation. Assays with antigen in the absence of sera served as negative controls.
8
DNA-Protein Interaction Analysis
9
Peptide-receptor binding assay protocol
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Unless otherwise specified, reagents were obtained from commercial sources. LC-MS grade water was purchased from Huberlab (Aesch, Switzerland); methanol and acetonitrile (both HPLC gradient grade) were purchased from VWR (Darmstadt, Germany). Ammonium formate and formic acid were purchased from Sigma-Aldrich (Buchs, Switzerland). Other solvents and reagents were purchased from Merck (Darmstadt, Germany). Fmoc-Arg(Pbf) Wang resin was obtained from Activotec (Cambridge, UK). Amino acids and coupling reagents were purchased from Iris Biotech (Marktredwitz, Germany). Recombinant human receptors and biotinylated human VEGF-A165 were purchased from R&D Systems (Minneapolis, MN, USA). Chemiluminescent, streptavidin-horseradish peroxidase conjugate and DPBS were obtained from Thermo Scientific (Waltham, MA, USA).
10
Extracellular Vesicle Aptamer Binding Assay
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For each assay, a 5′-biotinylated aptamer was incubated with extracellular vesicles in binding buffer that contained 100 µg/ml tRNA at room temperature for 2 h. The solution was passed through a nitrocellulose filter (EMD Millipore) and washed with 5 ml binding buffer. The filter was exposed to UV irradiation (254 nm) and was blocked in binding buffer that contained 3% BSA and 0.1% Tween-20 (Wako Pure Chemical Industries, Ltd.) for 60 min. Following washing, the filter was incubated with streptavidin-horseradish peroxidase conjugate (Thermo Fisher Scientific, Inc.; cat. no. N100; dilution, 1:1,000) for 60 min at room temperature and washed. Then, the Amersham ECL Prime Western Blotting Detection reagent (GE Healthcare Life Sciences, Chalfont, UK) was used to visualize the chemiluminescence of the 5′-biotinylated aptamer on the filter.
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