Recombinant proteins (200 ng) diluted in coating buffer (SeraCare, Milford, MA) were incubated in a 96-well microtiter plate (NUNC, Rochester, NY) overnight at 4°C. Then, the plate was blocked using 5% bovine serum albumin in phosphate-buffered saline (PBS) and incubated 1 h at room temperature, and subsequently washed four times in selection buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 5 mM KCl). The modified apMNK2F aptamers (apMNK2FT and apMNKQ2), conjugated with biotin, were diluted in selection buffer at the concentrations indicated in the figures, denatured for 10 min at 95°C and cooled for 10 min on ice. Next, 100 μL of the diluted aptamers were added to each well, the plate was incubated at 37°C for 1 h and washed four times with selection buffer to remove unbound ssDNA. Afterward, 100 μL of a 1/1,000 dilution of horseradish peroxidase-conjugated streptavidin (GE Healthcare, Madrid, Spain) were added to the individual wells. After 1 h incubation at 37°C on a shaking platform, the plates were washed four times and developed using ABTS solution (Roche, Madrid, Spain) according to the manufacturer’s instructions. OD 405 nm values were determined using a SpectraFluor microplate reader (TECAN, Barcelona, Spain).
Streptavidin conjugated horseradish peroxidase
Manufactured by GE Healthcare
Sourced in United States
Streptavidin-conjugated horseradish peroxidase is a biotechnological tool used in various laboratory applications. It combines the high-affinity binding of streptavidin to biotin with the enzymatic activity of horseradish peroxidase. This reagent is commonly used in immunoassays, blotting techniques, and other biochemical protocols that require the detection or quantification of biotinylated targets.
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Lab products found in correlation
96 well clear polystyrene high bind stripwell microplate, by Corning (2 mentions)
Abts solution, by Roche (1 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (1 mentions)
Coating buffer, by LGC (1 mentions)
Spectrafluor microplate reader, by Tecan (1 mentions)
Enhanced chemiluminescence detection reagent, by GE Healthcare (1 mentions)
Amplitaq gold polymerase, by Thermo Fisher Scientific (1 mentions)
Biotinylated nad, by Bio-Techne (1 mentions)
Western lightning chemiluminescence reagent kit, by PerkinElmer (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Immunosorp maxi plates, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by BD (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
7 protocols using streptavidin conjugated horseradish peroxidase
1
Recombinant Protein Binding Assay
2
Comprehensive HPV Genotyping Protocol
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An aliquot (5 μL) of the purified DNA was PCRamplified (total reaction volume 30 μL) with AmpliTaq Gold polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and biotinylated PGMY09/11 primers to amplify the L1 gene of mucosal HPVs. Biotinylated human leukocyte antigen (HLA) primers were used to amplify cellular HLA DNA. Positive (0.1 pg/mL of HPV16 full length genomic DNA in a plasmid) and negative controls (dH 2 O) were used to assess the sensitivity of PCR and detect contaminating HPV DNA in reagents. The PCR products (10 μL) were analyzed on 1.5% agarose gels to assess HPV and HLA DNA amplification; amplification of HLA DNA served as an internal control to confirm template integrity. Reverse blotting hybridization was performed as described (Unger et al. 2009) . Briefly, 15 μL of denatured PCR products were allowed to hybridize with oligonucleotide probes specific for 31 HPV genotypes (HPV6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 66, 68, 69, 70, 73, 82, 83, and 84) immobilized on a Biodyne C membrane (Pall corporation, Port Washington, NY, USA) using a Miniblotter MN45 (Immunetics, Cambridge, MA, USA). The hybridized DNA was detected using the horseradish peroxidase-conjugated streptavidin (GE Healthcare, Piscataway, NJ, USA) and the enhanced chemiluminescence detection reagent (GE Healthcare).
3
ADP-Ribosylation Assay for Actin
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Toxins were partially purified from culture supernatants by precipitation with 70% ammonium sulphate as described previously [6 (link)]. ADP ribosyltransferase assays were performed as previously described [48 (link)]. Briefly, precipitated supernatant protein (50 µg) was incubated for 60 minutes at 37°C with 10 µg of actin in assay buffer (20 mM Tris-HCl pH 7.5, 1 µM dithiothreitol (DTT), 40 µM ATP, 40 µM CaCl2, 5 µM MgCl2) and 10 µM of biotinylated NAD+ (Trevigen). The reaction was heat inactivated at 95°C for 5 minutes in 4x SDS sample buffer (240 mM Tris-Cl (pH 6.8), 40% glycerol (v/v), 8% SDS (w/v), 5% (v/v) 2-mercaptoethanol, 0.05% (v/v) bromophenol blue and separated by 10% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and biotinylated proteins were detected with horseradish peroxidase-conjugated streptavidin (GE Healthcare Life Sciences) and the Western Lightning Chemiluminescence reagent kit (Perkin-Elmer), following the manufacturer’s instructions. Relative band intensities were determined by densitometry using Image Lab Software (Bio-Rad). Data were analysed using GraphPad Prism 6 and statistical significance assessed using an unpaired t-test with a 95% confidence interval.
4
Quantifying Integrin Capture via ELISA
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Corning 96 Well Clear Polystyrene High Bind Stripwell Microplate (product #2592, Corning, Corning, NY, USA) were coated overnight with 5 μg ml−1 appropriate anti-integrin antibodies in 0.05 M Na2CO3 (pH 9.6) at 4 °C and were blocked in PBS containing 0.05% Tween-20 (PBS-T) with 5% BSA for 1 h at RT. Integrins were captured by overnight incubation of 50 μl cell lysate at 4 °C. Unbound material was removed by extensive washing with PBS-T, and wells were incubated with streptavidin-conjugated horseradish peroxidase (GE Healthcare) in PBS-T containing 1% BSA for 1 h at 4 °C. Following further washing, biotinylated integrins were detected by a chromogenic reaction with ortho-phenylenediamine.
5
OVA-Specific Antibody ELISA Protocol
6
Western Blot Analysis of Proteins
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Western blot analyses were performed to evaluate ANG II effects on the protein expression of D 1 R, OCTNs, and OCTs by using membrane preparations according to Kouyoumdzian et al. [23] (link). Protein concentration was determined according to Lowry assays [22] . Samples of membrane preparation were incubated with the following polyclonal antibodies from Santa Cruz Biotechnology (SCBT) (Dallas, Texas, USA): A secondary immunoreaction was performed with a corresponding biotinylated antibody (1 : 1500 dilution), followed by exposure to streptavidin-conjugated horseradish peroxidase (GE Healthcare Life Sciences, 1 : 2000 dilution). The samples were revealed by chemiluminescence, which was performed through incubation with an ECL reagent (Amersham Pharmacia Biotech) for 1-5 min. The density of the respective bands was quantified by densitometric scanning using a Hewlett-Packard scanner and ImageJ analyzer software (RSB). To prevent inaccuracies in protein loading, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured as an internal standard for each blot (rabbit polyclonal anti-GAPDH (FL-335) (sc-25778, SCBT, 1 : 1000 dilution). Protein levels were calculated and expressed as the ratio between the integrated optical densities corresponding to different proteins of interest and GAPDH.
7
Quantification of Biotinylated Receptors
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Corning 96 Well Clear Polystyrene High Bind Stripwell Microplate (product #2592, Corning) were coated overnight with 1 μg/ml of appropriate anti-VE-cadherin, NRP1 or transferrin receptor 1 antibodies in 0.05 M Na2CO3 (pH 9.6) at 4 °C and were blocked in PBS containing 0.01% Tween-20 (PBS-T) with 5% BSA for 1 h at RT. Proteins were captured by overnight incubation of 50 μl cell lysate at 4 °C. Unbound material was removed by extensive washing with PBS-T, and wells were incubated with streptavidin-conjugated horseradish peroxidase (GE Healthcare) in PBS-T containing 1% BSA for 1 h at 4 °C. Following further washing, biotinylated receptors were detected by a chromogenic reaction with ortho-phenylenediamine.
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