Enzyme-linked competition binding assay was performed as previously reported with a slight modification (14 (link), 24 (link)). Briefly, human plasma FN was coated on the high-bind plate (Corning 3590) overnight at 10 μg/mL of concentration. The biotinylated FUD and PEG-FUD (hereafter named as b-FUD and b-PEG-FUD, respectively) were prepared via manufacturer’s protocol using NHS-biotin-ester (Pierce). The FN-coated plate was blocked with 5% bovine serum albumin (BSA) in tris-buffered saline containing 0.05% Tween 20 (TBS-T). 0.5 nM of b-FUD or b-PEG-FUD was added to the plate simultaneously with different concentrations (1000, 100, 50, 10, 5, 1, 0 nM) of unlabeled FUD, PEGFUD, or PEG-mFUD in 0.1% BSA in TBS-T. After 2 h of incubation at RT and washing with TBS-T, alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch) was added at 1:20,000 for 1 h at RT. After washing, 100 uL of 1-step PNPP (Thermo Fisher) was added as a substrate, and absorbance was measured at 405 nm. The reaction was stopped using 2 N NaOH. The data was presented as the percent of b-FUD or b-PEG-FUD bound to the plate, where b-FUD and b-PEG-FUD were without a competitive inhibitor as a positive control. The binding affinity of Cy5- conjugates and NOTA- conjugates for adsorbed FN was confirmed using the same method described above.
1 step pnpp
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 1-step PNPP is a reliable and versatile laboratory reagent designed for a wide range of applications. It serves as a substrate for various enzymes, enabling the detection and quantification of enzymatic activity. The product provides a simple and efficient means to facilitate various analytical and research procedures.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Blocker casein, by Thermo Fisher Scientific (1 mentions)
High binding microplate, by Greiner (1 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Magellan 7, by Tecan (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
4 protocols using 1 step pnpp
1
Enzyme-linked competition binding assay for fibronectin
2
ELISA Assay for Protein Detection
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Soluble antigen was diluted to 2 μg/mL in Carbonate Coating Buffer (0.2 M Carbonate-Bicarbonate, pH 9.4 (Thermo Fisher)) and 20 μL was dispensed into the wells of a 384-well High Binding microplate (Greiner). The plate was sealed and incubated at 4°C overnight. After washing 3X with PBS + 0.1% Tween-20 (in house), the plate was blocked with 80 μL of Casein Blocker in PBS (Thermo Fisher) for 60 minutes. Following washing, 20 μL of normalized CHO supernatants at 5, 1.25, 0.31 and 0.08 μg/mL were added to the wells of the ELISA plate and incubated for 60 minutes. The expression supernatants derived from minipreps for both the standard primers and the rh-PCR Generation 2 primers were tested. After washing 3X with PBS + 0.1% Tween-20, 20 μL of 1:1000 dilution of Goat anti-Human kappa-AP (Southern Biotech) in Casein Blocker was added to each well and incubated 60 minutes. After washing again, 20 μL of 1-Step PNPP (Thermo Fisher) was added and the plate incubated until color developed. The plate was read on a Spectramax 384 Plus spectrophotometer at 405 nm.
3
Quantification of Antibody Isotypes and Anti-dsDNA
4
Serological Detection of Gonococcal Antigens
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ELISA plates (Immulon 4 HBX) were coated with 2 μg/mL of purified recombinant NGO0690, NGO0948, NGO1701 and native gonococcal PIB (PIB1B) [58 (link)] in carbonate buffer pH 9.0 (100 µL/well) or with formalin-fixed N. gonorrhoeae (1–1.5 × 108 CFU/mL) in PBS (100 µL/well) overnight at 4 °C. Plates were washed, and blocked with 1% BSA in PBS/0.05% Tween-20 (PBS/T) for 1 h at R.T. prior to overnight incubation at 4 °C with human sera as above (1:100 dilution), followed by incubation with an AP-conjugated secondary anti-human total IgG (Southern Biotech, Birmingham, AL, USA) and 1-step PNPP (p-nitrophenyl phosphate) reagent (Thermo Fisher Scientific). O.D.405 values were measured spectrophotometrically. Individual serum specimens were tested in triplicate and IgG levels expressed as the combined mean O.D.405 minus the O.D.405 of the control antigen without serum (referred to as blank throughout the Methods and Results sections) ± SD for each antigen.
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