Monolayers of MeWo cells (1 х 105 cell/cm2) were inoculated with 5 plaque forming unit (PFU) per cm2 of cell-associated pOka viruses for 2 hrs, followed by addition of medium, DMSO, or pimecrolimus (10 μM). The drug-containing medium was changed every 48 hrs before the cells were fixed with 4% PFA at 4 days post-infection. Immunohistochemistry (IHC) was performed on the fixed cells using mouse monoclonal anti-VZV antibody (Mixed, GeneTex #GTX38720, 1:2000 diluted in PBS), followed by incubation with biotinylated anti-mouse IgG (Vector Laboratories #BA-9200), and alkaline phosphatase streptavidin (Jackson ImmunoResearch #016-050-084). The plaques were visualized by staining with a mixture of FastRed salt (Sigma Aldrich) and Naphthol AS-MX phosphate (Sigma Aldrich). Images of plaques were taken with Axio microscope (Zeiss) using 2.5X lens. The plaques were outlined and the area of plaque was calculated using Image J. Monolayers of infected MeWo cells processed with IHC stain was also taken by BZ-X710 fluorescence microscope (Keyence) at 2X lens, and plaque frequency and total infected area were analyzed by ImageJ automatic particle counting. Experiments were performed at least three times.
Streptavidin alkaline phosphatase
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
Streptavidin-alkaline phosphatase is a conjugate used in various biotechnological and immunological applications. It consists of the streptavidin protein coupled to the enzyme alkaline phosphatase. The primary function of this conjugate is to provide a detection system that can be used to identify the presence of biotin-labeled targets in sample analyses.
Automatically generated - may contain errors
Lab products found in correlation
P nitrophenyl phosphate, by Merck Group (5 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (4 mentions)
Avidin hrp, by Thermo Fisher Scientific (4 mentions)
Folin reagent, by Bio-Rad (4 mentions)
Ige mouse uncoated elisa kit with plates, by Thermo Fisher Scientific (4 mentions)
Biotin conjugated rat anti mouse ige paired antibodies, by BD (3 mentions)
Mcpt 1 mmcp 1 mouse uncoated elisa kit with plates, by Thermo Fisher Scientific (3 mentions)
Mouse anti avi tag antibody, by GenScript (2 mentions)
Bsa standard, by Merck Group (2 mentions)
Alkaline copper tartrate, by Bio-Rad (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
Axio microscope, by Zeiss (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Fastred salt, by Merck Group (1 mentions)
Pnpp substrate, by Merck Group (1 mentions)
Elisa 96 well plates, by Corning (1 mentions)
Xmark microplate absorbance spectrophotometer, by Bio-Rad (1 mentions)
A7030, by Merck Group (1 mentions)
Mab6811, by R&D Systems (1 mentions)
19 protocols using streptavidin alkaline phosphatase
1
Quantifying Varicella-Zoster Virus Plaque Formation
2
Competitive Binding Assay for Anti-LPS Antibodies
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Human anti-LPS mAbs were biotinylated using the EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). Labeled mAbs were tested for binding to LPS by ELISA and the optimal concentration of each mAb to achieve 70% maximal binding was determined. Unlabeled mAbs were serially diluted and added to ELISA 96-well plates (Corning) coated overnight at 4 °C with 5 µg ml−1 LPS in PBS. After 1 h, biotinylated anti-LPS mAbs were added at the concentration achieving 70% maximal binding and the mixture was incubated at room temperature for 1 h. Plates were washed and antibody binding was revealed using Alkaline Phosphatase-streptavidin (Jackson Immunoresearch). After washing, pNPP substrate (Sigma-Aldrich) was added and plates were read at 405 nm. The percentage of inhibition was calculated as a percent decrease of signal vs. the maximum signal achieved with the labeled mAb.
3
Porcine IL-1β Quantification by ELISA
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Porcine IL-1β in the cell-free supernatants was determined using ELISA established in our lab previously [31 (link)]. Briefly, Immulon 2 HB U plates (#3655, Thermo Fisher) were coated with a mouse anti-porcine IL-1β antibody (MAB6811, R&D Systems) at 2 μg/mL in PBS overnight. The plates were blocked with 1% bovine serum albumin (BSA) (A7030, Sigma) in PBS for 1 h, and were incubated with either samples or the standard for 2 h. Two-fold serial dilutions of the recombinant porcine IL-1β protein (681-PI-010, R&D Systems) in diluent (0.1% BSA in Tris-buffered saline with 0.05% Tween 20) was used for the standard curve. Next, the plates were incubated with goat anti-porcine IL-1β biotinylated antibody (BAF681, R&D Systems) at 50 ng/mL in the diluent for 1 h, and further incubated for 1 h with alkaline phosphatase-streptavidin (016-050-084, Jackson ImmunoResearch, West Grove, PA, USA) that was diluted in a ratio of 1:5000 by the diluent. After incubation with 1 mg/mL of p-nitrophenyl phosphate in the diethanolamine buffer (1 M diethanolamine, 0.5 M MgCl2, pH 9.8), optical densities were measured at 405 nm with a reference at 490 nm using the xMark Microplate Absorbance Spectrophotometer (Bio-Rad).
4
Quantification of Oligoclonal IgM Bands
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Paired samples of serum and CSF were aliquoted and stored at −80°C until the assay was performed. OCMB were detected by isoelectric focusing (IEF) and immunodetection according to the technique described by Villar et al. (8 (link)). Briefly, serum samples were diluted in saline before the IEF in order to reach the same concentration range as in CSF samples. Focusing was performed on a Multiphor II Electrophoresis System (GE Healthcare, Chicago, USA) at pH 5 to 8. Then, proteins were transferred to a PVDF membrane by Western blot. Finally, immunodetection was performed by biotin-conjugated-goat anti-human IgM (Sigma) and streptavidin-alkaline phosphatase (Jackson ImmunoResearch).
5
Quantifying Antibody-Antigen Interactions via ELISA
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The 96-well plates were coated overnight at 4°C with mouse anti-Avi-tag antibody (Genscript) at 2 μg/ml in PBS. Plates were washed 4 times with PBS and 0.05% (vol/vol) Tween and blocked with 3% (wt/vol) bovine serum albumin (BSA) in PBS for 1 h. Concurrently, 5-fold serial dilutions from 50 μg/ml of rabbit or human mAbs were preincubated with 1 μg/ml of purified Avi-tagged SOSIP protein for 1 h. The mAb-SOSIP mixture was then transferred to the ELISA plate and incubated for 1 h. Plates were washed four times and incubated with 0.5 μg/ml of biotinylated mAb for 1 h and were washed again; binding was detected with streptavidin-alkaline phosphatase (Jackson Immunoresearch) at 1:1000 for 1 h. MAbs were biotinylated using the N-hydroxysuccinimide (NHS)-micro biotinylation kit (Pierce).
6
ELISPOT Assay for Anti-OVA Antibodies
7
ELISA Protocol for IgE and MCPT-1
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Biotin-conjugated rat anti-mouse IgE-paired antibodies were obtained from BD BioSciences (San Jose, CA, USA). Streptavidin alkaline phosphatase was obtained from Jackson ImmunoResearch (West Grove, PA, USA). BSA standard (at 2 mg/mL) was purchased from Sigma (St. Louis, MO, USA). p-nitro-phenyl phosphate was obtained from Sigma (St. Louis, MO, USA). Alkaline copper tartrate was purchased from BioRad (Hercules, CA, USA). Folin reagent was purchased from BioRad (Hercules, CA, USA). The following reagents were obtained from Invitrogen (Waltham, MA, USA): IgE Mouse Uncoated ELISA Kit with Plates; StreptAvidin-HRP, TMB substrate; MCPT-1 (mMCP-1) Mouse Uncoated ELISA Kit with Plates; Avidin-HRP, TMB substrate. Tissue Protein Extraction Reagent [T-PERTM, a proprietary detergent in 25 mM bicine and 150 mM sodium chloride (pH 7.6)] was from ThermoFisher Scientific (Waltham, MA, USA). A protease (serine, cysteine, acid proteases and aminopeptidases) inhibitor cocktail was obtained from Sigma-Aldrich (St. Louis, MO, USA).
8
Quantifying TG2 Transamidase Activity
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The TG2 transamidase activity assay was performed as previously described [62 (link)]. Briefly, 96-well plates were coated with 1 mg/mL N,N-dimethylated casein in PBS overnight at 4 °C. After washing with PBST, recombinant human TG2 (obtained from Zedira or in-house purified TG2) was incubated with 20 μM of the TG2 substrate 5BAPA (Thermo Fisher Scientific) and ACR or NC9 in the presence or absence of 5 mM CaCl2 in 50 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM EDTA, 5 mM DTT, and 5% glycerol for 1 h at 37 °C. Then, TG2-mediated incorporation of 5BAPA was quenched by the addition of 50 mM EDTA. After washing with PBST, immobilized 5BAPA was detected with streptavidin-alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) reacting with p-nitrophenyl phosphate (Sigma-Aldrich). Absorbance at 405 nm was measured using an EnSight Multimode Plate Reader (Perkin Elmer Inc.).
9
Characterization of Fcγ Receptor Ligands
10
Mouse IgE Quantification Assay
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The following chemicals and reagents were obtained from the sources indicated in parentheses: Biotin conjugated rat anti-mouse IgE paired antibodies and isotype standards (BD BioSciences, San Jose, CA, USA); para-Nitrophenylphosphate (Sigma, St Louis, MO, USA); streptavidin alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA, USA); protein estimation reagents: bovine serum albumin standard and reagents A and B (Sigma, St Louis, MO, USA). Pre-made SDS-PAGE gels were purchased from Bio-Rad (catalog #4561094). The cellulose membranes for Western blot were purchased from Bio-Rad (catalog #1620145); as were the molecular weight markers (Bio-Rad Precision Plus Protein catalog #161-0373; Thermo Scientific PageRuler Prestained Protein Ladder Product #26616); substrate buffer (Southern Biotech TMB membrane substrate catalog #0304-01); blocking buffer (5% BSA).
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