AF594 C5 Maleimide

Guidelines (The following are recommended experimental protocols for guidance only and require adjustment according to your specific needs)
1. Working Solution Preparation
1.1 Solvent: 1.5% (w/v) β-octyl glucoside (OG) buffer (25 mM MES, 25 mM K HEPES, 125 mM KCl, 1 mM EDTA, pH 6.7).
1.2 Notes: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Isolated native membrane protein (rhodopsin)^[1]:
3.2 Incubation conditions: Label the Cys³¹⁶ site of rhodopsin with AF594 C5 Maleimide at 20°C in the dark.

3.2 Sample type: Purified recombinant protein (ClyA mutant CC6/264 Q56C E252C, purified under reducing conditions)^[2]:
3.2 Incubation conditions: Incubate the protein with AF594 C5 Maleimide at a 1:1 stoichiometric ratio in 10 mM KH₂PO₄/K₂HPO₄ (pH 7.4) at 22°C for 4.5 h.
3.3 Washing step: Purify the dual-labeled oxidized ClyA mutant using a Superdex 200 gel filtration column equilibrated with PBS (pH 7.3).

4. Control Experiments
4.1 Set up a control experiment by mixing Rho-Alexa 488 and Rho-Alexa 594 at a 1:1 ratio in 1.5% OG micelles to verify the absence of significant FRET in monomeric rhodopsin.

5. Detection and Analysis
5.1 Instrument type: Fluorescence spectrophotometer equipped with excitation/emission monochromators; confocal fluorescence spectrometer for single-molecule Förster resonance energy transfer (FRET) detection.
5.2 Excitation/emission wavelengths: Excitation (Ex): 585 nm, 595 nm; Emission (Em): 620 nm.
5.3 Result analysis:
5.3.1 For isolated native membrane protein (rhodopsin): A decrease in acceptor emission intensity at 620 nm indicates enhanced dispersion of rhodopsin in the membrane; an increase in acceptor emission intensity at 620 nm indicates aggregation of rhodopsin; rhodopsin labeled with AF594 C5 Maleimide produces red fluorescence emission; the fluorescence is associated with the membrane (reconstituted in phospholipid bilayers).
5.3.2 For purified recombinant protein (ClyA mutant): Assess conformational changes of the labeled protein by measuring FRET efficiency; expected FRET efficiency values are: approximately 0.76 for the protomer state, approximately 0.46 for the monomer state, and approximately 0.15 for the disulfide-trapped intermediate state.