This dark red fluorescent ghost ring peptide conjugate selectively binds to F-actin and has much higher photostability than the fluorescein-ghost ring peptide conjugate. Peptidyl derivatives are often used in nanomolar concentrations and are used as convenient probes for labeling, identifying, and quantifying F-actin in formaldehyde-fixed and permeated tissue sections, cell cultures, or cell-free experiments. Ghost cyclin binds to the actin filament much more tightly than to the actin monomer, resulting in a decrease in the rate constant at which the actin subunits dissociate from the filament end, thereby essentially stabilizing the actin filament by preventing the filament from depolymerizing. Moreover, it was found that phoropeptide inhibited ATP hydrolysis activity of F-actin. The function of ghost pen cyclic peptide is different in different concentrations in cells. When introduced into the cytoplasm at low concentrations, porcinocyclic peptide absorbs the less polymerized cytoplasmic actin and fibrin into the stable island of the aggregated actin polymer, but it does not interfere with the stressed fibers, the thick bundles of microfilaments. The properties of porcine cyclic peptides are an effective tool to study the distribution of F-actin in cells by labeling porcine cyclic peptides with fluorescent analogues and using them to stain actin filaments for optical microscopy. Fluorescent derivatives of ghost cyclic peptides have been shown to be very useful in locating actin filaments in living or fixed cells, as well as in vitro observation of individual actin filaments. Fluorophosphorin derivatives have been used as important tools to study actin networks at high resolution.

A convenient probe often used to label, identify, and quantify F-actin in formaldehyde-fixed and permeated tissue sections, cell cultures, or cell-free experiments.

1. Add 30μLDMSO to powder vial.

2. Add 1 µL conjugated solution of phorus cyclic peptide SF 647 to 1 mL PBS containing 1% BSA.

Note 1: Unused reserve solutions of cyclopeptide conjugates should be equally divided and stored at -20 ℃, away from light.

Note 2: Different cell types may stain differently. The preparation of the concentration of the working solution of the conjugated cyclic peptide needs to be based on the actual situation.

3. Staining cells:

3.1 Formaldehyde fixation was performed, and cells containing 3.0-4.0% formaldehyde were incubated in PBS for 10-30 minutes at room temperature.

Note: Avoid using any fixatives that contain methanol, as methanol can destroy actin during the fixation process. The preferred fixative is formaldehyde without methanol.

3.2 Flush fixed cells with PBS 2-3 times.

3.3 Optional: Add 0.1% Triton X-100 to PBS to fix cells for 3 to 5 minutes to increase permeability. Flush the cells with PBS 2-3 times.

3.4 A 100μL/ well (96-well plate) working solution of the phlebocyclopeptide conjugate was added to the fixed cells and stained at room temperature for 20 to 90 minutes.

3.5 The cells are gently rinsed with PBS 2 to 3 times to remove excess ghost ring peptide conjugates before adding a cover glass, which is then performed under a microscope, sealed and imaged.

The sample was prepared in a microporous plate hole

Remove the liquid from the sample

Added ghost peptide SF 647 labeling solution (100μL/ well)

The cells were stained at room temperature for 20 to 90 minutes

Clean the cells and observe the sample under a microscope

Note: Heat the vial to room temperature and centrifuge briefly before opening.