The assay was performed as described in the AlphaLISA Kit (PerkinElmer). Briefly, 2 μl reaction products were incubated with 8 μl AlphaLISA Aβ1-40/42 Acceptor beads at 23°C for 1 hour. After another 30-minute incubation with 10 μl AlphaLISA Aβ1-40/42 Donor beads in the dark at 23°C, the samples were read using Envision-Alpha Reader (PerkinElmer). The readings were expressed in arbitrary unit.
Envision alpha reader
Manufactured by PerkinElmer
Sourced in United States
The Envision-Alpha Reader is a high-performance microplate reader designed for efficient fluorescence and luminescence detection. It provides accurate and reliable measurements for a wide range of assays, supporting both endpoint and kinetic analysis.
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Lab products found in correlation
8 protocols using envision alpha reader
1
AlphaLISA Assay for Amyloid-beta
2
Optimized Aβ Production Assay
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To get a better profile of Aβ production, we used C99-Flag as substrates and the concentration of transfected DNA was optimized by preliminary tests and set to 320 ng per well with 0.96 µl X-tremeGENE 9 Reagent. After 1 day of growth, the assay was performed as a standard two-step protocol, in which 5 μl cultured supernatant (or cell lysate) were incubated with 5 μl AlphaLISA Aβ1–40/42 acceptor beads and biotinylated anti-Aβ antibody at 23 °C for 1 h, followed by another 30-min incubation with 10 μl AlphaLISA Aβ1–40/42 donor beads in the dark at 23 °C. Photon counts were determined in 384 plates using an Envision-Alpha Reader (PerkinElmer, Waltham, MA, USA).
3
Quantitative Aβ40 and Aβ42 Assay with γ-Secretase
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Purified wild-type or T4 lysozyme fused γ-secretase, either in amphipols or digitonin, was mixed with APP-C99 in 0.2% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), 50 mM Hepes, pH 7.0, 0.1% phosphatidylcholine, and 0.025% phosphatidylethanolamine and incubated at 37 °C for 4 h as described (38 (link)). To detect the cleavage products Aβ40 and Aβ42, the AlphaLISA assay was performed following the standard protocol as described in the AlphaLISA kit (PerkinElmer). Briefly, 2-μL reaction samples were incubated at 22 °C for 1 h with 8 μL AlphaLISA Aβ1–40/42 Acceptor beads. After a 30-min incubation with 10 μL AlphaLISA Aβ1–40/42 donor beads in darkness at 22 °C, the samples were read by an Envision-Alpha Reader (PerkinElmer). The assay was repeated at least three times for each data point.
4
Amyloid-Beta Quantification by AlphaLISA
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AlphaLISA assay was conducted as described previously28 (link). HTL cells were transfected with 320 ng DNA using X-tremeGENE 9 Reagent (1 μg DNA:3 μL reagent). Media supernatants were collected the following day for Aβ42/Aβ40 detection. Briefly, 5 μL reaction samples were incubated at 23 °C with 5 μL AlphaLISA Aβ1–40/42 Acceptor beads and biotinylated anti-Aβ antibody for 1 h. After another 30-min incubation with 10 μL AlphaLISA Aβ1–40/42 donor beads in the dark at 23 °C, the samples were read in 384-well plates using an Envision-Alpha Reader (PerkinElmer).
5
Quantifying Protein-Nucleosome Interactions
6
Quantifying Protein-Nucleosome Interactions
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In vitro interactions between biotinylated nucleosomes and His-GFP tagged DNMT proteins were assessed by luminescence proximity AlphaScreen assay (PerkinElmer) as described40 (link). Briefly, 100 nM biotinylated nucleosomes and 100 nM His-tagged proteins were incubated with 5 μg/mL streptavidin-coated donor beads and 5 μg/mL nickel-chelated acceptor beads (PerkinElmer) in 100 μL total volume of AlphaScreen buffer (50 mM MOPS, pH7.4, 50 mM NaF, 50 mM CHAPS, and 0.1 mg/ml BSA) for 1.5 h in the dark at room temperature. Photon counts were determined in 384 plates using an Envision-Alpha Reader (PerkinElmer) (Extended Data Fig. 2a ). Each experiment was repeated at least four times. Data were validated for normality by Shapiro-Wilk and Kolmogorov-Smirnov tests and analyzed by one-way ANOVA using GraphPad Prism.
7
Quantifying Amyloid-beta Secretion in CHO Cells
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Chinese hamster ovary (CHO) cell lines overexpressing the human APP695 (CHO-APPWT) or the London mutated APP695 (CHO-APPV717L) were previously described [19 (link)]. CHO cells were grown in DMEM/F-12 1:1 medium, supplemented with 10% heat-inactivated fetal bovine serum, 0.2% Pen/Strep, 2% HT supplement and 300 µM Proline (Sigma, Kawasaki, Japan). One day before CHO supernatant media collection, a full-medium replacement was performed with BrainPhys. CHO cell conditioned BrainPhys medium was collected 24 h later, and Aβ concentrations were measured using Alpha-LISA kits specific for human Aβ1–X (AL288C, PerkinElmer, Waltham, MA, USA) and Aβ1–42 (AL276C, PerkinElmer). Briefly, the human Aβ analyte standard was diluted in the BrainPhys medium. For the assay, 2 µL of cell culture medium or standard solution was added to an Optiplate-384 microplate (PerkinElmer). A volume of 2 µL of the 10X mixture, including acceptor beads and the biotinylated antibody, was then added to the wells with culture media or standard solution. Following incubation at room temperature for 1 h, 16 µL of 1.25X donor beads was added to the respective wells and incubated at room temperature for 1 h. Luminescence was measured using an EnVision-Alpha Reader (PerkinElmer) at 680 nm excitation and 615 nm emission wavelengths. Control treatment was performed using unconditioned/blank BrainPhys medium.
8
SMCHD1-TET Protein Interaction Assay
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AlphaScreen (PerkinElmer) assays were performed for determining the in vitro interaction between biotin-SMCHD1 and His-tagged TET proteins following the manufacturer’s protocol. Briefly, 200 nM SMCHD1-biotin and 200 nM TET2FL-His or TET2-CD-His were incubated at room temperature for 1 hour. The protein sample was then incubated with streptavidin-coated donor beads (final concentration of 10 μg/ml) and nickel-chelate acceptor beads (final concentration of 10 μg/ml) in a total volume of 100 μl of AlphaScreen buffer containing 50 mM MOPS (pH 7.4), 50 mM NaF, 50 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and BSA (0.1 mg/ml) for 1 hour in the dark at room temperature. The photon counts were detected in 384-well plates by the EnVision Alpha reader (PerkinElmer).
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