His-tagged CRBN-DDB1 protein was prepared following the procedure reported by Matyskiela et al(51 (link)) and GST-tagged LCK (1–509) protein was acquired from GeneTex. The assay mixture contained 240 nM His-tagged CRBN-DDB1, 60 nM GST-tagged LCK (1–509), 20 μg/mL nickel chelate AlphaLISA acceptor, and glutathione donor beads (PerkinElmer, # 6765300) and serially diluted test compounds in a buffer comprising 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% BSA, 0.05% tween20, and 0.005% proclin-300. A 96-well plate containing 5× test compound, 5× His-tagged CRBN/DDB1, and 5× GST-tagged LCK was incubated at rt for 1 h. After the incubation, 5 μL of the solution was transferred to a 384-well white AlphaPlate (PerkinElmer) in duplicate followed by 10 μL nickel chelate AlphaLISA acceptor (1:100) and 10 μL glutathione donor beads (1:100). The plate was sealed and mixed on a MixMate (Eppendorf) for 60 min at room temperature and then luminescence detection was collected on an Envision plate reader (PerkinElmer).
Glutathione donor beads
Manufactured by PerkinElmer
Sourced in United States
Glutathione donor beads are a type of laboratory equipment used in biochemical assays and research applications. They provide a source of reduced glutathione, an important antioxidant and cofactor in various cellular processes. The beads are designed to efficiently deliver the glutathione to the desired experimental system.
Automatically generated - may contain errors
Lab products found in correlation
Nickel chelate acceptor beads, by PerkinElmer (3 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
Alphaplate, by PerkinElmer (1 mentions)
Nickel chelate alphalisa acceptor, by PerkinElmer (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Mixmate, by Eppendorf (1 mentions)
Pherastar plate reader, by BMG Labtech (1 mentions)
Enspire 2300 multimode reader, by PerkinElmer (1 mentions)
Alphalisa universal buffer, by PerkinElmer (1 mentions)
4 protocols using glutathione donor beads
1
Quantification of CRBN-DDB1:LCK Interaction
2
AlphaScreen Assay for SIX1-EYA1 Interaction
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The AlphaScreen assay was performed using GST-SIX1 and His-EYA1 proteins in the same small molecule pool as described previously.[19 (link)] In brief, equal amounts (7.5 μL) of GST-SIX1 and His-EYA1 proteins were mixed with 5 μL glutathione donor beads and 5 μL nickel chelate acceptor beads (PerkinElmer, Waltham, MA, USA, #6760603M). After 30 min, equal volumes (2 μL) of individual compounds were added into the protein mixture and incubated at 16°C for 2 h. The AlphaScreen signals were collected by reading plates in an Envision Multilabel Reader (PerkinElmer, #2105-0010), and compounds that caused signal values to decrease significantly (<5000) were selected as candidates.
3
AlphaScreen Protein-Protein Interaction Assay
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AlphaScreen was conducted with 10 μg/mL of glutathione donor beads (PerkinElmer, Waltham, MA) and nickel chelate acceptor beads (PerkinElmer) in 10 mM Bis-Tris (pH 7.0), 1 mM TCEP, 0.02 % casein, and 0.1 % Tween-20. Compounds were plated first followed by addition of one the proteins at 600 nM and incubation for 30 min. The second protein was added and incubated for 30 min, then the beads were added simultaneously and the final mixture was incubated for 2 h. Experiments were conducted in 384-well plates (6007290; PerkinElmer) and results were read on a PHERAstar plate reader (BMG Labtech, Cary, NC).
4
Probing MLL1 Interactions with Chromatin Complexes
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To study the interaction of MLL1 with the RBBP5/ASH2L or WDR5/RBBP5/ASH2L complexes with AlphaScreen assays, 0.5 μm His‐tagged RBBP5 protein, 0.5 μm His‐tagged ASH2L protein and (if needed) 0.5 μm His‐tagged WDR5 were pre‐incubated for 30 min at 4 °C to form the corresponding complexes. A 10 μL aliquot of the complexes was loaded in each well of a microplate (1/2 Area plate™‐96; PerkinElmer). Then, 10 μL of 0.5 μm GST‐tagged MLL1‐SET was added and incubated for 1 h at 22 °C. Afterward, 0.8 μg nickel‐chelate acceptor beads (PerkinElmer) and 0.8 μg glutathione donor beads (PerkinElmer) were added and incubated for another 1 h in the dark at 22 °C. As negative controls, empty beads or beads incubated with 0.5 μm GST protein were included. The AlphaScreen light signal was measured with an EnSpire™ 2300 Multimode reader (PerkinElmer). The experiments were conducted in AlphaLISA Universal Buffer (PerkinElmer AL001C) containing PBS (10 mm phosphate, 137 mm NaCl, 2.7 mm KCl) pH 7.2, 0.1% BSA, and 0.01% Proclin‐300.
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