Ghost pen cyclic peptide-AMCA labeled probes selectively bind to F-actin. Used in nanomolar concentrations, porcinocyclic peptide derivatives are convenient probes for labeling, identifying, and quantifying F-actin in formaldehyde-immobilized and permeated tissue sections, cell cultures, or cell-free experiments. Actin is a globular, approximately 42kDa protein that is found in almost all eukaryotic cells. It is also one of the highly conserved proteins, differing no more than 20% from algae and different species of humans. Actin is the monomer subunit of two filaments in the cell: microfilaments, one of the three major components of the cytoskeleton, and microfilaments, which are part of the contractile apparatus in muscle cells. Thus, actin is involved in many important cellular processes, including muscle contraction, cell movement, cell division and cytoplasmic division, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell connections and cell shape.

Ghost cyclin binds to the actin filament more tightly than the actin monomer, resulting in a reduced rate constant for the actin subunits to dissociate from the filament-like end, essentially stabilizing the actin filament by preventing the filament from depolymerizing. In addition, it was found that ghost cyclin inhibited the ATP-hydrolyzing activity of F-actin. Ghost pen cyclic peptide acts differently in different concentrations in cells. When introduced into the cytoplasm at low concentrations, porcinocyclic peptide aggregates less polymerized forms of cytoplasmic actin as well as filamentins into a stable island of aggregated actin polymers, but it does not interfere with the stressed fibers, the thick bundles of microfilaments. The properties of ghost ring peptides are a useful tool for studying the distribution of F-actin in cells by labeling ghost ring peptides with fluorescent analogues and staining actin filaments with them for optical microscopy. Fluorescent derivatives of ghost cyclic peptides have proven to be very useful in locating actin filaments in living or fixed cells, as well as in visualizing individual actin filaments in vitro. Fluorophosphorin derivatives have been used as important tools in the study of high resolution actin.

Label, identify, and quantify F-actin in formaldehyde-immobilized and permeated tissue sections, cell cultures, or cell-free experiments

1. Add 1 μL of the porcinocyclic peptide-AMCA conjugate solution to 1 mL of 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 concentration of the working solution of the conjugated cyclopeptide should be prepared accordingly.

2. Staining cells:

2.1 Perform formaldehyde fixation. 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.

2.2 The fixed cells were washed with PBS 2-3 times.

2.3 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.

2.4 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.

2.5 The cells are gently rinsed with PBS 2 to 3 times to remove excess ghost ring peptide conjugates before the addition of 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 in the plate

AMCA solution was added (100μL/ well)

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

Clean the cells to examine the sample under a microscope

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