HPsensor 2

Guide
(The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: For most dyes, an organic solvent such as anhydrous DMSO is typically used for dissolution.
1.2 Concentration recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Distilled water adjusted to pH 8 using 0.1 M NaOH; Carmody buffer is used for pH-dependent experiments.
2.2 Working concentration: 2 μM for general protein staining; 0.5 μM for surface hydrophobicity and binding affinity measurements; 1X, 3X, 10X relative to protein concentration for native PAGE experiments.
Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Purified protein samples (bovine serum albumin (BSA), myoglobin (Mb), apomyoglobin (ApoMb), beta-lactoglobulin (β-lg)).[1]
3.2 Incubation conditions:
3.2.1 For general protein staining: Incubate 2 μM HPsensor 2 with 2 μM protein (1:1 ratio) at room temperature for 1 hour.
3.2.2 For surface hydrophobicity and binding affinity measurements: Incubate 0.5 μM HPsensor 2 with protein samples at 25 °C for 1 hour.
3.2.3 For pH-dependent experiments: Incubate 0.5 μM HPsensor 2 with 0.1 μM heated/unheated protein in Carmody buffer (pH 3, 5, 7, or 9) at 25 °C for ~30 minutes.
3.2.4 For native PAGE: Incubate 2 μg protein with 1X, 3X, or 10X HPsensor 2 at room temperature for 1 hour.

4. Controls
4.1 Set up free dye control (HPsensor 2 without protein) to measure background fluorescence.
4.2 Set up protein-only control to subtract protein auto-fluorescence from experimental readings.
4.3 Set up ANS-stained protein control for comparative fluorescence signal analysis.

5. Detection & Analysis
5.1 Instrument: Fluorometer for fluorescence spectroscopy; gel imaging system for native PAGE analysis.
5.2 Ex/Em wavelength: Excitation at 528 nm, emission at 579 nm.
5.3 Result analysis:
5.3.1 Fluorescence intensity changes: HPsensor 2 shows weak fluorescence in aqueous solution, with significant fluorescence enhancement (up to 60-fold compared to ANS) when bound to hydrophobic protein surfaces (highest for BSA, followed by ApoMb, lowest for Mb).
5.3.2 Fluorescence localization: Binds to exposed hydrophobic surfaces of proteins.
5.3.3 Native PAGE results: HPsensor 2 exhibits stronger UV signal than ANS for protein bands, with signal intensity correlating with protein surface hydrophobicity.
5.3.4 pH and heat effect: Reduced fluorescence signal is observed at low pH and for heated (denatured) proteins, except for β-lg at pH 9.
5.3.5 Binding affinity: Dissociation constants (Kd) are 1.2 μM for Mb, 0.33 μM for ApoMb, and 0.034 μM for BSA.
5.3.6 Surface hydrophobicity (S0): Calculated as the slope of net relative fluorescence intensity vs protein concentration, with values of 2934 for Mb, 65212 for ApoMb, and 658608 for BSA.