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Streptavidin conjugated donor beads

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Streptavidin-conjugated donor beads are a type of luminescent bead designed for use in Alphascreen and AlphaLISA assays. They are coated with streptavidin, a protein that binds to biotin-labeled molecules. These beads can be used as a donor component in proximity-based assays to detect and quantify biomolecular interactions.

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Lab products found in correlation

Protein a conjugated acceptor beads, by PerkinElmer (4 mentions) Envision plate reader, by PerkinElmer (2 mentions) Envision 2105 multimode plate reader, by PerkinElmer (1 mentions) Gsh acceptor beads, by PerkinElmer (1 mentions) Alphalisa acceptor beads, by PerkinElmer (1 mentions) Enspire microplate reader, by PerkinElmer (1 mentions)

Close spelling variants of this product

Streptavidin-conjugated ALPHA donor beads (4 citations)

6 protocols using streptavidin conjugated donor beads

1

In Vitro Gamma-Secretase Activity Assay

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In vitro γ-secretase activity assay (NICD production level) was carried as described previously42 and illustrated (ExtFig 2i). Briefly, to detect NICD production by the cleavage of γ-secretase, HEK293-NotchΔE cells were lysed with 0.25% CHAPSO after siRNA transfection. 5μl of lysates were incubated with 20μl detection buffer (Protein A-conjugated acceptor beads (PerkinElmer), NICD antibody (SM320), 9E10 anti-human c-myc conjugated to biotin antibody and streptavidin-conjugated donor beads (PerkinElmer)) for overnight at room temperature. NICD levels were measured as AlphaLISA signals by EnVision Plate Reader (PerkinElmer).
To detect NICD production by the cleavage of γ-secretase in IFITM3 KO cells, cell membranes were lysed with 0.25% CHAPSO and incubated with a recombinant Notch substrate, N1-Sb1 at 37 °C for 2 hrs. 20 μl of lysates were incubated with 20 μl detection buffer (Protein A-conjugated acceptor beads (PerkinElmer), NICD antibody (SM320, generated in our lab) and streptavidin-conjugated donor beads (PerkinElmer)) for overnight at room temperature42 . NICD levels were measured as described above.
Hur J.Y., Frost G.R., Wu X., Crump C., Pan S.J., Wong E., Barros M., Li T., Nie P., Zhai Y., Wang J.C., TCW J., Guo L., McKenzie A., Ming C., Zhou X., Wang M., Sagi Y., Renton A.E., Esposito B.T., Kim Y., Sadleir K.R., Trinh I., Rissman R.A., Vassar R., Zhang B., Johnson D.S., Masliah E., Greengard P., Goate A, & Li Y.M. (2020). Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease. Nature, 586(7831), 735-740.
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2

In Vitro Gamma-Secretase Activity Assay

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In vitro γ-secretase activity assay (NICD production level) was carried as described previously42 and illustrated (ExtFig 2i). Briefly, to detect NICD production by the cleavage of γ-secretase, HEK293-NotchΔE cells were lysed with 0.25% CHAPSO after siRNA transfection. 5μl of lysates were incubated with 20μl detection buffer (Protein A-conjugated acceptor beads (PerkinElmer), NICD antibody (SM320), 9E10 anti-human c-myc conjugated to biotin antibody and streptavidin-conjugated donor beads (PerkinElmer)) for overnight at room temperature. NICD levels were measured as AlphaLISA signals by EnVision Plate Reader (PerkinElmer).
To detect NICD production by the cleavage of γ-secretase in IFITM3 KO cells, cell membranes were lysed with 0.25% CHAPSO and incubated with a recombinant Notch substrate, N1-Sb1 at 37 °C for 2 hrs. 20 μl of lysates were incubated with 20 μl detection buffer (Protein A-conjugated acceptor beads (PerkinElmer), NICD antibody (SM320, generated in our lab) and streptavidin-conjugated donor beads (PerkinElmer)) for overnight at room temperature42 . NICD levels were measured as described above.
Hur J.Y., Frost G.R., Wu X., Crump C., Pan S.J., Wong E., Barros M., Li T., Nie P., Zhai Y., Wang J.C., TCW J., Guo L., McKenzie A., Ming C., Zhou X., Wang M., Sagi Y., Renton A.E., Esposito B.T., Kim Y., Sadleir K.R., Trinh I., Rissman R.A., Vassar R., Zhang B., Johnson D.S., Masliah E., Greengard P., Goate A, & Li Y.M. (2020). Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease. Nature, 586(7831), 735-740.
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3

TR-FRET Assay for BRD Protein Interactions

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TR-FRET assay kits from BPS Bioscience (San Diego, CA) that were specific for BRD2 BD1 (Cat# 31022), BRD2 BD2 (Cat# 32522), BRD3 BD1 (Cat# 32513), BRD3 BD2 (Cat# 32523), BRD4 BD1 (Cat# 32613), and BRD4 BD2 (Cat# 32617) were used. The BRD ligands were diluted with water. A master mix consisting of 1× BRD Homogeneous Assay Buffer and diluted BRD ligands was prepared. The BRD proteins were thawed on ice and diluted with 1× BRD Homogeneous Assay Buffer. Subsequently, 1.5 μL master mix was added to each well of a microplate (Cat# LP-0200; Labcyte) and reactions were initiated by adding 5 μL diluted BRD protein to each well. The plate was incubated at room temperature for 30–60 min. GSH Acceptor beads (Cat#AL109C) and Streptavidin-conjugated donor beads (Cat# 6760002S) (both from PerkinElmer, Waltham, MA) were diluted with 1× BRD Homogeneous Detection Buffer 1, after which a 10-μL acceptor bead mixture was added to each well. After incubation at room temperature for 30 min, a 10-μL donor bead mixture was added to each well, followed by incubation at room temperature for 15–30 min. Alpha-counts were read with an EnVision 2105 multimode plate reader (Cat# 2105-0010, PerkinElmer).
Jeong S., Kim H.R., Shin J.H., Son M.H., Lee I.H, & Roe J.S. (2023). Streamlined DNA-encoded small molecule library screening and validation for the discovery of novel chemotypes targeting BET proteins. Molecular Therapy. Nucleic Acids, 32, 637-649.
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4

Notch Cleavage Assay Protocol

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Cells were seeded in 96-well culture plates. Media was then removed, and cells were washed with PBS. Cleavage assay mixture included a final concentration of PIPES Buffer (50 mM PIPES, pH 7.0, 150 mM KCl, 5 mM CaCl2, 5 mM MgCl2), 0.25% CHAPSO detergent, protease inhibitor cocktail, Notch substrate (0.4 μM), and 0.1% DMSO or JC2 at 1 μM final concentration. Cleavage assay was performed at 37 °C for 2.5 h. Product of Notch cleavage was recognized by AlphaLISA detection comprised of anti-activated Notch antibody SM320, protein A-conjugated acceptor beads, and streptavidin-conjugated donor beads (PerkinElmer) [66 (link)]. Activity readout was expressed as arbitrary units minus the background signal from γ-secretase inhibitor sample and normalized to protein concentration.
Ledo J.H., Liebmann T., Zhang R., Chang J.C., Azevedo E.P., Wong E., Silva H.M., Troyanskaya O.G., Bustos V, & Greengard P. (2020). Presenilin 1 phosphorylation regulates amyloid-β degradation by microglia. Molecular Psychiatry, 26(10), 5620-5635.
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5

Notch Cleavage Assay Protocol

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Cells were seeded in 96-well culture plates. Media was then removed, and cells were washed with PBS. Cleavage assay mixture included a final concentration of PIPES Buffer (50 mM PIPES, pH 7.0, 150 mM KCl, 5 mM CaCl2, 5 mM MgCl2), 0.25% CHAPSO detergent, protease inhibitor cocktail, Notch substrate (0.4 µM), and 0.1% DMSO or JC2 at 1 µM final concentration. Cleavage assay was performed at 37 °C for 2.5 h. Product of Notch cleavage was recognized by AlphaLISA detection comprised of anti-activated Notch antibody SM320, protein A-conjugated acceptor beads, and streptavidin-conjugated donor beads (PerkinElmer) [66 (link)]. Activity readout was expressed as arbitrary units minus the background signal from γ-secretase inhibitor sample and normalized to protein concentration.
Ledo J.H., Liebmann T., Zhang R., Chang J.C., Azevedo E.P., Wong E., Silva H.M., Troyanskaya O.G., Bustos V, & Greengard P. (2020). Presenilin 1 phosphorylation regulates amyloid-β degradation by microglia. Molecular Psychiatry, 26(10), 5620-5635.
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6

Quantification of TFPIα Levels

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Anti-K2 was conjugated to AlphaLISA acceptor beads (Perkin Elmer). Anti-K1, for measurement of total TFPI, and anti-K3C, for measurement of TFPIα, were conjugated with biotin. Plasma (20μL, diluted in HBSA containing 0.1% Tween-20) and 20 μL of a mixture containing the conjugated acceptor bead (12.5μg/mL) and biotinylated anti-K1 (2.5 nM) or anti-K3C (5 nM) was added to the well of a 96-well plate and incubated (1 hr, 37°C). Streptavidin-conjugated donor beads (Perkin Elmer) were added (25 μL, 20 μg/mL) and incubated (1 hr, RT). The plate was read on an Enspire microplate reader (Perkin Elmer) and the results compared to a TFPIα standard curve.
Wood J.P., Baumann Kreuziger L.M., Ellery P.E., Maroney S.A, & Mast A.E. (2017). Reduced prothrombinase inhibition by tissue factor pathway inhibitor contributes to the factor V Leiden hypercoagulable state. Blood Advances, 1(6), 386-395.
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