Cy5 maleimide (Cyanine5 maleimide) is a highly sensitive anatase dye for the detection of free thiols in natural protein extracts.

Instructions for use
I. Solution preparation
1. Stock solution: Dissolve Cy5 Maleimide in an appropriate solvent, such as dimethylsulfonamide (DMSO) or dimethylformamide (DMF). Common stock solution concentration: 1–10 mM. Store at -20°C and protect from light to prevent photodegradation.
2. Working solution: Dilute the stock solution to the required concentration (e.g. 1–50 µM) using an appropriate buffer (e.g. PBS or HEPES buffer, pH 7. Prepare freshly when used to ensure optimal labeling.
II. Thiol labeling steps using Cy5 Maleimide
1. Protein sample preparation:
1) Extract proteins from cells or tissues using an appropriate protein extraction buffer (e.g. RIPA buffer or PBS).
2) Ensure that there are no other groups in the protein sample that may react with Cy5 Maleimide (e.g. amino or carboxyl groups) to avoid interfering with the labeling process.
2. Labeling reaction:
1) Mix Cy5 Maleimide with protein sample in an appropriate buffer (e.g., PBS, pH 7.4).
2) Incubate at room temperature for 30 min to 1 h to allow the dye to react with free thiol groups in the protein.
3) The typical molar ratio of Cy5 Maleimide to protein is 1:1 to 10:1, depending on the desired labeling level.
3. Termination and purification:
1) After the reaction is complete, terminate the labeling reaction by adding a thiol-containing reagent such as dithiothreitol (DTT) or tris(2-aminoethyl)phosphine (TCEP).
2) Purify the labeled protein to remove excess dye using methods such as dialysis, size exclusion chromatography, or affinity purification.
4. Cell and imaging applications
Cell imaging:
(1) Labeling of live or fixed cells by using biomolecules labeled with Cy5 Maleimide or directly in SPAAC/CuAAC labeling.
(2) Wash cells with PBS to remove unbound dye.
(3) Analyze fluorescence signals using confocal or fluorescence microscopy, and select a filter set suitable for Cy5 (excitation wavelength: 646 nm, emission wavelength: 662 nm).
Tissue imaging:
(1) Stain tissue sections with Cy5 Maleimide-labeled probes.
(2) Wash sections thoroughly to remove background fluorescence.
(3) Analyze using a fluorescence microscope or whole-slice imaging system.
In vivo imaging:
(1) Inject Cy5 Maleimide-labeled probes into animal models.
(2) Perform near-infrared fluorescence imaging using an in vivo imaging system (such as IVIS) or similar platform.
(3) Monitor the distribution, targeting, and clearance of labeled probes.
Notes
1. Photosensitivity: Cy5 Maleimide is light-sensitive and should be avoided from long-term exposure to light and stored in a light-proof environment.
2. Reaction optimization: Optimize dye concentration, reaction time, and buffer conditions according to experimental conditions to achieve efficient labeling and reduce background.
3. Copper toxicity (for CuAAC): Copper ions may affect biological samples, so the copper concentration should be carefully controlled, or SPAAC reaction should be selected due to copper toxicity issues.
4. Stability: The stock solution should be aliquoted to avoid repeated freezing and thawing to maintain the activity of the solution. "