Recombinant human His-tagged ATAD2A (produced in-house), biotinylated H4 K5Ac (1-25) peptide, and test compound were added to a 384-well OptiPlate (Perkin Elmer) and incubated at room temperature for one hour. The assay buffer consisted of 50 mM HEPES, pH 7.5, 100 mM NaCl, and 0.12 mM Triton X-100. Final concentrations for this reaction were as follows: 100 nM His-ATAD2, 100 nM peptide, variable concentrations of compound (3-fold serial dilutions), and 1% (v/v) DMSO. Streptavidin donor beads and nickel chelate acceptor beads, both from a Perkin Elmer AlphaScreen Histidine Detection Kit, were then added to the OptiPlate to a final concentration of 10 μg/mL each. Following a 2 hour incubation at room temperature, the microplate was read on an Envision Plate Reader (Perkin Elmer). Percent of control (POC) values were calculated from the following formula: POC = sample signal-average background signal / average maximum signal – average background signal * 100. The average maximum signal was obtained from wells containing all assay components except test compound. The average background signal pertained to wells with all assay components except ATAD2A and test compound.
Alphascreen histidine detection kit
Manufactured by PerkinElmer
The AlphaScreen Histidine Detection Kit is a versatile laboratory tool used for the detection and quantification of histidine-tagged proteins. It utilizes AlphaScreen technology, a proximity-based assay that generates a luminescent signal when the target histidine-tagged protein is bound to the kit's components. The kit provides a sensitive and reliable method for researchers to monitor and analyze histidine-tagged proteins in various applications.
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Lab products found in correlation
7 protocols using alphascreen histidine detection kit
1
ATAD2A histone acetyltransferase inhibitor assay
2
AlphaScreen™ Macrodomain Binding Assay
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The AlphaScreen™ reactions were carried out in 384-well plates (Alphaplate, PerkinElmer, Waltham, MA) in a total volume of 40 μL in buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 0.5 mM TCEP, 0.1% BSA, and 0.05% CHAPS. All reagents were prepared as 4X stocks and 10 μL volume of each reagent was added to a final volume of 40 μL. All compounds were transferred acoustically using ECHO 555 (Beckman Inc) and preincubated after mixing with purified His-tagged macrodomain protein (250 nM) for 30 min at RT, followed by addition of a 10 amino acid biotinylated and ADP-ribosylated peptide [ARTK(Bio)QTARK(Aoa-RADP)S] (Cambridge peptides) (625 nM). After 1 h incubation at RT, streptavidin-coated donor beads (7.5 μg/ml) and nickel chelate acceptor beads (7.5 μg/mL); (PerkinElmer AlphaScreen™ Histidine Detection Kit) were added under low light conditions, and plates were shaken at 400 rpm for 60 min at RT protected from light. Plates were kept covered and protected from light at all steps and read on BioTek plate reader using an AlphaScreen™ 680 excitation/570 emission filter set. For counter screening of the compounds, 25 nM biotinylated and hexahistidine-tagged linker peptide (BIO-6His) (PerkinElmer) was added to the compounds, followed by addition of beads as described above.
3
Peptide-Chemokine Binding Assay
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The AlphaScreen® Histidine detection kit was purchased from PerkinElmer (6760619M lot 2457886), and the assay was set up in white-bottomed Proxiplate™ 384 Plus microplates (PerkinElmer) following the manufacturer's instructions. The assay buffer used was 50 mm HEPES, 150 mm NaCl, 0.1% BSA, 0.01% Tween 20, 1% DMSO, pH 7.5, and the final volume in each well was 20 μl. Briefly, biotinylated chemokine (recombinant; final concentrations, 1.25 nm (CCL8, produced in-house), 5 nm (CCL2, Almac), and 2.5 nm (CCL3, Almac) was preincubated at room temperature for 15 min with different concentrations of each peptide. His-tagged P672 (final concentrations, 2.5 nm (CCL8), 5 nm (CCL2), and 1.25 nm (CCL3)) was then added to each well, and the plate was incubated at room temperature for 30 min. Finally, acceptor and donor beads were added as a 1:1 suspension in buffer to each well, and the plate was further incubated at room temperature for 1 h. The data were obtained by reading the plate using a Pherastar FSX plate reader (excitation, 680 nm; emission, 570 nm) and was analyzed using GraphPad Prism.
4
AlphaScreen Assay for ADP-ribosylation
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The AlphaScreen reactions were carried out in 384-well plates (Alphaplate, PerkinElmer, Waltham, MA) in a total volume of 40 μL in buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 0.5 mM TCEP, 0.1% BSA, and 0.05% CHAPS. All reagents were prepared as 4X stocks and 10 μL volume of each reagent was added to a final volume of 40 μL. All compounds were transferred acoustically using ECHO 555 (Beckman Inc) and preincubated after mixing with purified His-tagged macrodomain protein (250 nM) for 30 min at RT, followed by addition of a 10 amino acid biotinylated and ADP-ribosylated peptide [ARTK(Bio)QTARK(Aoa-RADP)S] (Cambridge peptides) (625 nM). After 1h incubation at RT, streptavidin-coated donor beads (7.5 μg/mL) and nickel chelate acceptor beads (7.5 μg/mL); (PerkinElmer AlphaScreen Histidine Detection Kit) were added under low light conditions, and plates were shaken at 400 rpm for 60 min at RT protected from light. Plates were kept covered and protected from light at all steps and read on BioTek plate reader using an AlphaScreen 680 excitation/570 emission filter set. For data analysis, the percent inhibition was normalized to positive (DMSO + labeled peptide) and negative (DMSO + macrodomain + peptide, no ADPr) controls. The IC50 values were calculated via four-parametric non-linear regression analysis constraining bottom (=0), top (=100), & Hillslope (=1) for all curves.
5
FGF19 and FGF21 Peptide Binding Assay
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FGF19 and FGF21 C-terminal WT and mutant peptides were custom synthesized and purified (>95% purity) (LifeTein LLC, South Plainfield, NJ). Binding of FGF19 and FGF21 peptides to β-Klotho was assessed using an AlphaScreen® assay. Briefly, 20 nM of β-Klotho ECD 6 × His, varying amounts of FGF19 and 21 peptides, and 40 nM of biotinylated human FGF19 or FGF21 protein were prepared in AlphaLISA universal buffer (PerkinElmer) and added to individual wells in 384-well white opaque plates (Greiner Bio-One). Subsequently, streptavidin donor beads and nickel chelate (Ni-NTA) acceptor beads (AlphaScreen Histidine detection kit; PerkinElmer) were diluted to 40 µg/mL and added to the plates. Total reaction volume was 8 µL. Plates were incubated for 3 hours at room temperature protected from light and read on the EnVision Multilabel Plate Reader (PerkinElmer) according to the manufacturer’s instructions. Assays were performed in triplicate. IC50 values were determined from a three-parameter logistic regression model with the least squares fit using GraphPad Prism version 7.
6
Screening for Anti-mTfR Nanobodies
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To select anti-mTfR nanobodies, two rounds of in solution selections were performed with 100 and 50 nM biotinylated mTfR (50741-M07H-100, Sino Biological), respectively. After the second round the library was subcloned into an expression vector (pBDS100, a modified pHEN6 vector with an OmpA signal peptide and a C-terminal 3xFlag/6xHis tag) [46 (link)]. The expression library was used to transform TG1 E.coli after which nanobodies were expressed from single colonies. These nanobodies were screened for direct binding to the biotinylated mTfR using the AlphaScreen Histidine Detection Kit (6760619 M, Perkin Elmer). The hits were sequenced and clustered according to sequence homology. One representative of each sequence cluster was recloned into the NT vector (pBDS100 with C-terminal NT), expressed and purified following the protocol by Pardon et al. [45 (link)]. In total, 7 nanobodies were successfully recloned and expressed.
7
Quantifying Bromodomain Interactions
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Recombinant His tagged BAZ2A and BAZ2B bromodomains were tested by alpha technology in the presence of a biotinylated histone H3 acetylated lysine 14 peptide (H3K(Ac)14), H-YQTARKSTGGK(Ac)APRKQLATKAK(Biotin)-OH) [26] using the AlphaScreen Histidine detection kit (Perkin Elmer). The assays were performed in 384 optiplates (Perkin Elmer) as already described [27] using equimolar amount of ligands at hooking point (750 nM) evaluated after 1 hr incubation at room temperature in buffer A (50 mM HEPES pH 7.4, 100 mM NaCl, 0.1% BSA, 0.05% CHAPS). Compounds, dissolved in DMSO, were tested in duplicate or dose-response and IC50 values were obtained by nonlinear regression of log(dose)response fit using GraphPad Prism software. DMSO was kept below 2% as final concentration and data were normalized against corresponding DMSO controls.
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