FRET-based nucleosomes sliding

Epidyne Fret Assay

Materials:

EpiDyne-FRET nucleosome remodeling substrate (EpiCypher 16-4201)

10X Assay Buffer 

200 mM Tris pH 7.5

500 mM KCl

30 mM MgCl₂

1.0 mg/mL BSA

ATP (Invitrogen Catalog No. PV3227) 

384w white proxiplate (Perkin Elmer 6008280)

Recombinant protein (diluted to 400nM in 1X remodeling buffer, stored in aliquots in -80 °C; or IP’d protein)

          ACF (EpiCypher 15-1013)

          SMARCA4 (EpiCypher 15-1014)

          SMARCA2 (EpiCypher 15-1015)

          5x Remodeling Assay Buffer (100 mM Tris HCl, pH 7.5, 250 mM KCl, 15 mM MgCl₂, 0.05% (w/v) BSA, 0.05% (v/v) Tween 20) 

Integra Voyager multichannel repeat pipetter

Tecan M1000 (or any 384-well Fluorescence microplate reader capable of Cy3/Cy5 FRET detection)

Methods*:

1. Determine the amount of recombinant protein (10-20 nM final, or max amount/rxn from IP), EpiDyne-FRET (20 nM final), and ATP (1-2 mM final) needed - based number of reactions with and without ATP (no ATP reactions are required as appropriate control for ATP-stimulated activity), treatments, replicates, etc. 

2. Prepare 2X recombinant protein solution (if final conc will be 20 nM prepare 40 nM solution in 1X remodeling buffer).

3. Prepare reaction components: 1) master mix of recombinant protein, substrate and assay buffer; 2) ATP solution or H2O for no ATP control reactions. Scale up master mix for total number of reactions + 1 extra reaction. 

4. Add 8 uL master mix per well to microplate using Integra Voyager multichannel repeat pipetter.

5. Add 2 uL ATP solution or H2O to appropriate wells using Integra Voyager multichannel repeat pipetter.

6. Immediately read plate in Tecan M1000 

Kinetic assay (duration 1 hr, kinetic interval 3 min)

Cy3 fluorescence intensity

Excitation: 535 nm, bandwidth 20nm 

Emission: 579 nm, bandwidth 20nm

Mode: top

Gain: manual 50

Flashes: mode 1[400 Hz] 50, settle time 0 ms

Z-position: manual 24648um 

TRF settings: 0us lag time, 20us integration time

Cy3 fluorescence intensity

Excitation: 535 nm, bandwidth 20nm 

Emission: 685 nm, bandwidth 20nm

Mode: top

Gain: manual 50

Flashes: mode 1[400 Hz] 50, settle time 0 ms

Z-position: manual 24648um 

TRF settings: 0us lag time, 20us integration time

*Modified from page 2 of Epicypher’s technical note:

https://www.epicypher.com/content/documents/technotes/EpiDyne_FRET.pdf

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