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Streptavidin xlent

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Streptavidin-XLent! is a high-quality streptavidin reagent that enables the detection and isolation of biotinylated molecules. It is designed to provide efficient and reliable performance in various bioassays and research applications.

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Lab products found in correlation

Heparanase, by R&D Systems (2 mentions) Biotin heparan sulfate eu cryptate, by PerkinElmer (2 mentions) Spectramax id5 microplate reader, by Molecular Devices (1 mentions) Phosphatase inhibitor cocktail, by Roche (1 mentions) Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Protein g sepharose 4 ff, by GE Healthcare (1 mentions) Spectramax i3x microplate reader, by Molecular Devices (1 mentions)

5 protocols using streptavidin xlent

1

Homogeneous TR-FRET Assay for PD1/B7-H1 Inhibition

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Example 12

The assay described is a homogenous TR-FRET assay using HTRF® assay technology requiring no wash steps. To a Costar 3676 microtitre plate 5 μl/well of biotinylated PD1/Fc at 1 nM diluted into PBS was added. This was followed with the addition of 5 μl/well streptavidin XLent (CisBio) at 4 nM diluted into assay buffer (PBS+0.1% BSA+0.8M KF). 5 μl/well of a titration of sample material diluted in PBS was added to relevant wells. For the definition of total binding, 5 μl of PBS or relevant sample buffer was added per well. To define non-specific binding, an excess (600 nM) of unlabelled B7H1/Fc or PD1/Fc was used. The final process was the addition of 5 μl/well of cryptate labelled B7H1/Fc (cryptate label—CisBio, B7H1/Fc-RnD Systems) diluted 1:100 into assay buffer. The assay plate was left for 3 hours at room temperature before being read on a HTRF® compatible plate reader.

Example of IC50 determinations in the human PD1/human B7-H1 ligand inhibition assay for anti-B7-H1 antibodies are provided in Table 19. All anti-human B7-H1 antibodies are in the IgG1-TM format.

TABLE 19
Example of IC50 determination (n = 2) in the human
PD1/human B7-H1 ligand inhibition assay for anti-B7-H1 IgG
ArithmeticStandard
AntibodymeanDeviation
2.9D101.1E−103.6E−11
2.7A4OPT1.6E−101.4E−10
2.14H9OPT1.1E−107.5E−11

US10400039B2. Targeted binding agents against B7-H1 (2019-09-03). MEDIMMUNE LIMITED [GB]. Inventors: Christophe Queva [US], Michelle Morrow [GB], Scott Hammond [US], Marat Alimzhanov [US], John Babcock [CA], Ian Nevin Foltz [CA], Jaspal Singh Kang [CA], Laura Sekirov [CA], Melanie Boyle [GB], Matthieu Chodorge [GB], Ross A. Stewart [GB], Kathleen Ann Mulgrew [US].
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2

Heparanase Activity Assay Protocol

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42 μL of HS trisaccharide solution in Milli-Q water (0.0038–500 μM) or just Milli-Q water (as a control), and 42 μL of heparanase (5.3 nM, R&D Systems) solution in pH 7.5 triz buffer (consisting of 20 mM TrisHCl, 0.15 M NaCl and 0.1% CHAPS) or just buffer as blank were added into microtubes and pre-incubated at 37 °C for 10 min bringing the [heparanase] to 0.5 nM. Next, 84 μL of biotin-heparan sulfate-Eu cryptate (Cisbio, Cat #: 61BHSKAA) (58.6 ng in pH 5.5 0.2 M NaOAc buffer) was added to the microtubes, and the resulting mixture was incubated for 60 min at 37 °C. The reaction mixture was stopped by adding 168 μL of Streptavidin-XLent! (Cisbio, Cat #: 611SAXLA) (1.0 μg/ml) solution in pH 7.5 dilution buffer made of 0.1 M NaH2PO4, 0.8 M KF, 0.1% BSA. After the mixture had been stirring at room temperature for 15 min, 100 μL (per well) of the reaction mixture was transferred to a 96 well microplate (Corning #3693 96 well, white polystyrene, half-area) in triplicates and HTRF emissions at 616 nm and 665 nm were measured by exciting at 340 nm using a SpectraMax iD5 Microplate Reader (Molecular Devices).
Zhu S., Li J., Loka R.S., Song Z., Vlodavsky I., Zhang K, & Nguyen H.M. (2020). Modulating Heparanase Activity: Tuning Sulfation Pattern and Glycosidic Linkage of Oligosaccharides. Journal of medicinal chemistry, 63(8), 4227-4255.
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3

Homogenous TR-FRET Assay for PD1/B7H1 Interaction

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Example 12

The assay described is a homogenous TR-FRET assay using HTRF® assay technology requiring no wash steps. To a Costar 3676 microtitre plate 5 μl/well of biotinylated PD1/Fc at 1 nM diluted into PBS was added. This was followed with the addition of 5 μl/well streptavidin XLent (CisBio) at 4 nM diluted into assay buffer (PBS+0.1% BSA+0.8M KF). 5 μl/well of a titration of sample material diluted in PBS was added to relevant wells. For the definition of total binding, 5 μl of PBS or relevant sample buffer was added per well. To define non-specific binding, an excess (600 nM) of unlabelled B7H1/Fc or PD1/Fc was used. The final process was the addition of 5 μl/well of cryptate labelled B7H1/Fc (cryptate label—CisBio, B7H1/Fc—RnD Systems) diluted 1:100 into assay buffer. The assay plate was left for 3 hours at room temperature before being read on a HTRF® compatible plate reader.

Example of IC50 determinations in the human PD1/human B7-H1 ligand inhibition assay for anti-B7-H1 antibodies are provided in Table 19. All anti-human B7-H1 antibodies are in the IgG1-TM format.

TABLE 19
Example of IC50 determination (n = 2) in the human
PD1/human B7-H1 ligand inhibition assay for anti-B7-H1 IgG
ArithmeticStandard
AntibodymeanDeviation
2.9D101.1E−103.6E−11
2.7A4OPT1.6E−101.4E−10
2.14H9OPT1.1E−107.5E−11

US11518809B2. Targeted binding agents against B7-H1 (2022-12-06). MEDIMMUNE LIMITED [GB]. Inventors: Christophe Queva [US], Michelle Morrow [GB], Scott Hammond [US], Marat Alimzhanov [US], John Babcock [CA], Ian Foltz [CA], Jaspal Singh Kang [CA], Laura Sekirov [CA], Melanie Boyle [GB], Matthieu Chodorge [GB], Ross A. Stewart [GB], Kathleen Ann Mulgrew [US].
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4

Profiling LSD1 Demethylase Activity

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Cortical or hippocampal tissues were isolated from the mice administered with compounds and were frozen-stored until the assay. The control tissues were isolated from the rats at 2 hours after vehicle treatment. Tissues were homogenized in radioimmunoprecipitation assay buffer (20-188, Millipore) containing protease inhibitor cocktail (4693132001, Roche) and phosphatase inhibitor cocktail (4906837001, Roche) and were precleared by Protein G Sepharose 4FF (17-0618-01, GE Healthcare). Then, LSD1 protein in the homogenate was immunoprecipitated by using anti-LSD1 antibody (2139, Cell Signaling Technology) and Protein G Sepharose 4FF, according to the manufacturer’s instructions. Histone demethylase activity in the immunoprecipitates was measured by HTRF detection system, using biotin-labeled monomethyl-H3K4 peptide (Scrum Inc.), europium cryptate-labeled anti–histone H3 antibody (64CUSKAZ, Cisbio Bioassays), and Streptavidin-XLent! (611SAXLB, Cisbio Bioassays), and the remaining demethylase activity was determined by HTRF ratio (time-resolved fluorescence at 665 nm/time-resolved fluorescence at 615 nm).
Baba R., Matsuda S., Arakawa Y., Yamada R., Suzuki N., Ando T., Oki H., Igaki S., Daini M., Hattori Y., Matsumoto S., Ito M., Nakatani A, & Kimura H. (2021). LSD1 enzyme inhibitor TAK-418 unlocks aberrant epigenetic machinery and improves autism symptoms in neurodevelopmental disorder models. Science Advances, 7(11), eaba1187.
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5

Heparanase Activity Assay Protocol

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A total of 42 μL of inhibitor solution in Milli-Q water (0.00016–4000 μM) or just Milli-Q water (as a control) and 42 μL of heparanase (5.3 nM, R&D Systems) solution in pH 7.5 tris buffer (consisting of 20 mM Tris HCl, 0.15 M NaCl, and 0.1% CHAPS) or just buffer as a blank were added into microtubes and preincubated at 37 °C for 10 min. Next, 84 μL of biotin-heparan sulfate-Eu cryptate (Cisbio, Cat # 61BHSKAA; 58.6 ng in pH 5.5 0.2 M NaCH3CO2 buffer) was added to the microtubes, and the resulting mixture was incubated for 60 min at 37 °C. The reaction mixture was stopped by adding 168 μL of Streptavidin-XLent! (Cisbio, Cat # 611SAXLA; 1.0 μg/mL) solution in pH 7.5 dilution buffer made of 0.1 M NaPO4, 0.8 M KF, and 0.1% BSA. After the mixture had been incubating at room temperature for 15 min, 100 μL (per well) of the reaction mixture was transferred to a 96-well microplate (Corning #3693 96-well, white polystyrene, half area) in triplicates and HTRF emissions at 616 and 665 nm were measured by exciting at 340 nm using SpectraMax i3x Microplate Reader (Molecular Devices).
Sletten E.T., Loka R.S., Yu F, & Nguyen H.M. (2017). Glycosidase Inhibition by Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymers. Biomacromolecules, 18(10), 3387-3399.
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