AF532 C5 Maleimide

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: DMSO[2].
1.2 Concentration recommendation: 0.5 μM VASP-AF532[1].

2. Working Solution Preparation
2.1 Diluent: KMEI buffer[1]; phosphate buffer (50 mM potassium phosphate, pH 7.4, 100 mM NaCl)[2].
2.2 Working concentration: 0.5 μM VASP-AF532[1].
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type descriptions:
3.1.1 Supported lipid bilayers (SLBs) with pre-immobilized β1 integrin tails, talin-1, and kindlin-2[1].
3.1.2 Protein nanocapsules (E279C mutant, engineered with surface cysteines)[2].
3.2 Incubation conditions:
3.2.1 For SLBs with pre-immobilized β1 integrin tails, talin-1, and kindlin-2: Incubate with 0.5 μM VASP-AF532 at room temperature[1].
3.2.2 For E279C protein nanocapsules: React at room temperature[2].
3.3 Washing steps:
3.3.1 For SLBs with pre-immobilized β1 integrin tails, talin-1, and kindlin-2: Wash 2 times with 0.8 mL KMEI buffer after incubation[1].
3.3.2 For E279C protein nanocapsules: Remove excess dye and reducing agents using Zeba desalting columns (40 kDa MWCO) with phosphate buffer as the exchange solution[2].

4. Controls

5. Detection & Analysis
5.1 Instrument type: Epifluorescence microscopy[1]; fluorescence detection instrument[2].
5.2 Ex/Em wavelengths: Corresponding to Alexa Fluor 532 (AF532)[2].
5.3 Result analysis:
5.3.1 For SLBs with pre-immobilized β1 integrin tails, talin-1, and kindlin-2:
5.3.1.1 VASP-AF532 alone does not induce phase separation or β1 integrin tail clustering.
5.3.1.2 Uniform fluorescence signal is observed across the SLB when VASP-AF532 is incubated alone.
5.3.2 For E279C protein nanocapsules:
5.3.2.1 Fluorescence intensity changes: Higher fluorescence intensity is observed for AF532-conjugated E279C nanocapsules compared to wild-type E2 nanocapsules.
5.3.2.2 Fluorescence localization: Fluorescence is localized on the external surface of the protein nanocapsules.
5.3.2.3 Color changes: Red-orange fluorescence is emitted from the conjugated dye.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.