ATTO 488 carboxylic acid

Operating Instructions (The following is a recommended experimental protocol for reference only, and adjustments should be made according to actual requirements)

1. Stock Solution Preparation
1.1 Solvent: Anhydrous dimethyl sulfoxide (DMSO).
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: Phosphate-buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂SO₄, 1.8 mM KH₂PO₄, pH 7.4); alternatively, 20 mM HEPES buffer at pH 8.3 can be used.
2.2 Working Concentration: 1 nM for fluorescence correlation spectroscopy (FCS) experiments; 0.5-30 μM for single-molecule total internal reflection fluorescence (TIRF) imaging.
2.3 Notes: Adjust the working solution concentration as needed; prepare it immediately before use.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Protein Samples (BSA-labeled): Suitable for labeling proteins containing primary amines. BSA and ATTO 488 NHS ester are incubated at room temperature for 2 h in 20 mM HEPES buffer (pH 8.3) at a molar ratio of 1:1, with the N-terminal amino group being the main target (lysine residues may also be labeled). Unreacted dye is removed using a Centrispin-10 size exclusion column (5 kDa cutoff).
3.1.2 Cell Samples (Adherent Cells): U2OS cells are incubated with Lysosome-488 probe (a conjugate of Atto 488 NHS ester and cathepsin-targeting peptide) for 30 min, followed by a 4 h post-incubation in growth medium to reduce background fluorescence.
3.1.3 Liposome/Supported Lipid Bilayer Samples: 1 nM Atto 488 is incubated with liposomes of different concentrations in PBS before FCS measurement; 0.5-30 μM Atto 488 is incubated on DOPC-supported lipid bilayers (SLB) in the dark for approximately 10 min, followed by rinsing with PBS at least 3 times.
3.2 Incubation Conditions
3.2.1 Protein Labeling: Room temperature, 2 h, 20 mM HEPES pH 8.3.
3.2.2 Cell Staining: 37 °C, 5% CO₂, 30 min probe incubation + 4 h post-incubation in medium.
3.2.3 SLB Imaging: Room temperature, dark condition, approximately 10 min.
3.3 Washing Procedures
3.3.1 Protein Labeling: Unreacted dye is removed by centrifugation using a Centrispin-10 size exclusion column (5 kDa cutoff).
3.3.2 Cell Samples: A 4 h post-incubation in growth medium is performed to reduce background.
3.3.3 SLB Samples: Rinse with PBS at least 3 times

4. Control Experiments
4.1 Unstained/Free Dye Control: FCS measurement of free Atto 488 (1 nM) in PBS is conducted to obtain the diffusion time and triplet state parameters of the free dye.
4.2 Covalent Binding Control: The same operation is carried out without the NHS linker. No single-molecule signal is detected after rinsing, which confirms that the protein binds via covalent bonds rather than non-specific adsorption.
4.3 pH Control: The pH of 100 nM ATTO 488 in MilliQ water is adjusted with HCl/NaOH, and the change in fluorescence intensity is measured to rule out the direct effect of pH on the fluorescence of the dye itself.
4.4 Colocalization Control: LysoTracker Green (50 nM) and GFP-LAMP1 are used as standard lysosome markers, and their colocalization with Lysosome-488 yields Pearson's coefficients of 0.776 and 0.811, respectively.

5. Detection and Analysis
5.1 Instrument Types: Fluorescence spectrophotometer, confocal microscope, TIRF microscope, SIM super-resolution microscope, fluorescence correlation spectroscopy (FCS) system.
5.2 Excitation/Emission Wavelengths: Excitation wavelength is 488 nm (alternatively 480 nm or 473 nm); emission wavelength is 524 nm, or detection is performed using a 505 nm long-pass filter / BLP01-488 long-pass + HQ 520/66 band-pass filter; excitation/emission slits are 2 nm.
5.3 Result Analysis
5.3.1 Fluorescence Intensity Changes: In the pH-responsive PAA brush system, fluorescence intensity is high at pH 3 (brush collapsed) and low at pH 10 (brush extended), with a switching amplitude of up to 80%, which is reversible and rapid. The decay of the FCS curve slows down after Atto 488 binds to liposomes; the Kp value is 253 in DOPC, 298 in DOTAP, and 73.7 in DOPG.
5.3.2 Fluorescence Localization: Lysosome-488 specifically labels lysosomes in U2OS cells, with Pearson's coefficients of 0.776 for colocalization with LysoTracker Green and 0.811 for colocalization with GFP-LAMP1; cell viability is >85%. The adsorption density of Atto 488 on DOPC-supported lipid bilayers is 30-40 molecules/10⁴ μm, indicating weak interaction with the membrane.
5.3.3 Color Change: Green fluorescence. No significant changes in fluorescence lifetime and quantum yield are observed before and after binding to liposomes.

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