T-sCPY-s500R-H

Operation Guide (The following is a recommended experimental protocol for guidance purposes only and requires adjustment according to your specific needs)
1. Stock Solution Preparation
1.1 Solvent: anhydrous DMSO.
1.2 Recommended concentration: 0.5-5 mM.
2. Working Solution Preparation
2.1 Diluent: cell culture medium containing 0.1% Pluronic F-127.
2.2 Working concentration: 50 nM; 500 nM.
2.3 Notes: adjust the working concentration as needed; prepare fresh before use.
3. Staining Procedure
3.1 Applicable to adherent cells:
3.1.1 Sample types: HEK293T, HeLa, U2OS cells, and U2OS cells expressing HaloTag-NLS.
3.1.2 Trypsin digestion is not specified as required.
3.2 Incubation conditions: incubate cells with T-sCPY-s500R-H at nanomolar concentrations (50 nM; 500 nM) for 12 h to overnight; protection from light is not specified as required.
3.3 Washing steps: direct labeling without washing is possible; or optionally wash twice with complete medium.
4. Control Setup
4.1 In vitro protein labeling experimental controls:
4.1.1 eDHFR-Halo protein alone (without probe, negative control).
4.1.2 T-sCPY-s500R-H probe alone (without protein, blank control).
4.2 Cell imaging controls:
4.2.1 Cells not transfected with eDHFR-Halo (negative control, to verify probe specificity)
5. Detection and Analysis
5.1 Instrument types: confocal microscope, microplate reader, fluorescence spectrophotometer.
5.2 Excitation/emission wavelengths:
5.2.1 Live-cell imaging: excitation Ex = 488 nm; donor (green) emission = 500-580 nm; acceptor (red) emission = 600-700 nm.
5.2.2 In vitro fluorescence detection: excitation Ex = 490 nm.
5.3 Result analysis:
5.3.1 Fluorescence intensity changes: binding of T-sCPY-s500R-H to HaloTag or eDHFR-Halo leads to enhanced green fluorescence; NADPH binding triggers a decrease in green fluorescence intensity (I₅₃₅) and an increase in red fluorescence intensity (I₆₃₅), thereby raising the FRET ratio (I₆₃₅/I₅₃₅); addition of NADPH further modulates fluorescence intensity.
5.3.2 Fluorescence localization: in unmodified adherent cells, fluorescence localizes to the cytoplasm; in U2OS cells expressing HaloTag-NLS, fluorescence localizes to the nucleus.
5.3.3 Color changes: donor Rhodamine produces green fluorescence; after NADPH stimulation, acceptor Rhodamine produces red fluorescence.
5.3.4 Protein labeling experiment: after the probe binds to eDHFR-Halo, SDS-PAGE fluorescence visualization detection shows a fluorescent band at ~50 kDa.

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