Prodan

Guidelines (The following are recommended experimental protocols for guidance only, and need to be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvents: Acetonitrile^[2], ethanol^[3], or DMF^[4] are optional.

2. Working Solution Preparation
2.1 Diluents: Optional options include 50 mM sodium phosphate buffer containing 0.1 M NaCl (pH 7.0, universal type), 25 mM sodium phosphate buffer containing 50 mM NaCl (pH 7.0, for SNP genotyping)^[1], water^[2], or buffer containing 5 mM phosphate, 20 mM KCl, 1 mM EDTA, pH 8.0 supplemented with 0.2 mM DTT^[4].
2.2 Working concentration: 0-5 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Oligodeoxynucleotide (ODN) samples (single-stranded or double-stranded ODNs)^[1]:
3.1.1.1 Incubation conditions: Incubate 2.5 μM single-stranded ODN containing PDNX with 2.5 μM complementary ODN at room temperature to form double strands; perform fluorescence detection at 25 °C.
3.1.2 Large unilamellar vesicles (LUVs) composed of phospholipids (lecithin, DOPC, or their mixtures) with optional cholesterol addition^[2]:
3.1.2.1 Incubation conditions: Hydrate dried lipid membranes with 2 μM Prodan aqueous solution at 25°C for approximately 5 min; for vesicles derived from electrospun membranes, dissolve 10 mg of membrane in 5 mL water containing Prodan, and stir to induce self-assembly.
3.1.3 Multilamellar vesicle (MLV) dispersions of symmetric saturated diacylphosphatidylcholines (CnPCs) with acyl chain lengths n=12-15 and 19-22^[3]:
3.1.3.1 Incubation conditions: Mix Prodan stock solution with CnPC stock solution, remove the solvent under vacuum, add water, then vortex/sonicate at a temperature slightly above the main transition temperature of each CnPC bilayer to prepare MLV dispersions.
3.1.4 Purified dimeric spectrin solution^[4]:
3.1.4.1 Incubation conditions: Incubate with 0.5 μM to 1 μM Prodan working solution at 25°C.

4. Control Setup
4.1 ODN samples^[1]:
4.1.1 Set single-stranded ODN containing PDNX as a control to distinguish double-stranded samples.
4.2 Lipid vesicle samples^[2]: Prepare control samples with the same phospholipid concentration but without Prodan for subtracting background absorption and fluorescence.
4.3 Spectrin samples^[4]: Use aqueous buffer containing only Prodan but no spectrin as a blank control to detect the fluorescence of unbound Prodan.

5. Detection and Analysis
5.1 Instrument types: Fluorescence spectrophotometer, fluorescence microplate reader (for SNP genotyping), etc.
5.2 Excitation/Emission Wavelengths
5.2.1 ODNs containing PDNX^[1]:
5.2.1.1 PDNU: 380 nm/524 nm (free nucleoside), 389 nm/520 nm (single-stranded ODN), 404 nm/522 nm (matched double strand); base-selective detection uses 450 nm excitation/520 nm emission; SNP genotyping uses 440±10 nm excitation filter/510±10 nm emission filter.
5.2.2 Lipid LUV samples^[2]: Excitation at 350 nm or 370 nm; monitor emission at 434 nm, 445 nm, 465 nm, 490 nm, and 525 nm.
5.2.3 Lipid MLV samples^[3]: Excitation at 361 nm; monitor emission in the range of 400 nm to 600 nm.
5.2.4 Spectrin samples^[4]: Excitation at 360 nm; measure emission at 430 nm and 520 nm.
5.3 Result Analysis
5.3.1 ODN samples^[1]:
5.3.1.1 Under 450 nm excitation, the fluorescence intensity of fully matched Watson-Crick double strands is significantly higher than that of single-stranded ODNs and mismatched double strands.
5.3.1.2 The Prodan fluorophore anchors to the major groove surface of the DNA double strand.
5.3.1.3 Fully matched double strands have a smaller Stokes shift value (lower wavenumber) compared with single-stranded ODNs and mismatched double strands.
5.3.2 Lipid LUV samples^[2]:
5.3.2.1 The emission band blue-shifts from 530 nm in aqueous solution to approximately 498 nm, and an emission band at 434 nm appears as the vesicle concentration increases.
5.3.2.2 The emission band at 434 nm corresponds to PRODAN localized in the deep nonpolar region of the lipid bilayer.
5.3.2.3 PRODAN exhibits triexponential decay in lecithin LUVs (corresponding to the water-bilayer interface, bilayer nonpolar region, and deep nonpolar core of the bilayer, respectively); it exhibits biexponential decay in pure DOPC LUVs (corresponding to the polar interface and bilayer nonpolar region, respectively).
5.3.2.4 Prodan in LUVs derived from electrospun membranes shows a red edge excitation shift (REES) of up to 8 nm when the excitation wavelength changes from 350 nm to 400 nm, indicating that it is in a motion-restricted microenvironment.
5.3.3 Lipid MLV samples^[3]:
5.3.3.1 Prodan localizes in regions of the bilayer corresponding to phase states, and the shift of the emission minimum correlates with changes in bilayer polarity.
5.3.3.2 Fluorescence second-derivative spectra show characteristic minima specific to bilayer phases: 430-440 nm for the bilayer gel phase (Lβ' or Pβ'), 480-490 nm for the liquid crystalline phase (Lα), and approximately 500 nm for the interdigitated gel phase (LβI).
5.3.3.3 Stack the second-derivative spectra obtained at every 1°C to construct a three-dimensional (3D) spectral imaging map for visualizing the change in bilayer phase state with temperature.
5.3.4 Spectrin samples^[4]:
5.3.4.2 Compared with unbound Prodan in aqueous buffer, the emission intensity of Prodan bound to spectrin increases by 2-fold; the fluorescence polarization degree rises from 0.05 in the unbound state to 0.27 in the bound state.
5.3.4.3 Prodan binds to the hydrophobic sites of spectrin.
5.3.4.4 The emission maximum blue-shifts by 87 nm (from 520 nm for unbound Prodan to 433 nm for Prodan bound to spectrin).