5-IAF is a fluorescein derivative of indoleacetamide, a fluorescent probe that can be used to label proteins and others containing free thiols (cysteine side chains). 5-IAF can label the catalytic (alpha) subunit of dog kidney Na,K-ATPase.

Instructions
1. Preparation and storage
a. Solubility:
5-IAF is usually provided in powder form and is easily soluble in DMSO, DMF or aqueous solution at pH ≥ 8. Recommended stock solution concentration: 10–50 mM (adjust according to experimental needs).
Before use, dissolve it in anhydrous solvent and aliquot into small volumes to avoid repeated freezing and thawing.
b. Storage conditions:
Powder: Store in a dry environment, away from light at -20°C or lower.
Solution: Short-term storage at 4°C, long-term storage at -20°C away from light is recommended.
2. Thiol labeling
a. Protein reduction treatment (if necessary): To ensure that the thiol groups are in a reduced state, protein samples can be pretreated with DTT or TCEP.
Use a thiol-free buffer (such as PBS or HEPES), pH 7–8 is the optimal range.
b. Labeling reaction:
Dilute the stock solution of 5-IAF to an appropriate working concentration (usually 0.1–1 mM). Add to the protein solution (usually 1–10 mg/mL) and mix gently.
c. Incubate at room temperature for 30 minutes to 2 hours, the specific time needs to be optimized according to the experiment.
d. Termination reaction: Terminate the labeling reaction by adding excess reducing agent (such as β-mercaptoethanol) or dilution.
e. Purification: Remove unbound 5-IAF and reaction byproducts using dialysis, gel filtration, or centrifugal ultrafiltration.
3. Fluorescence detection
Excitation wavelength: ~495 nm. Emission wavelength: ~520 nm (green fluorescence).
The fluorescent signal of the labeled protein can be detected in a fluorescence microscope or fluorescence spectrometer.