Rhodamine-Phalloidin (C09-1015-615)

This orange fluorescent ghost pen cyclic peptide conjugate (equivalent to TRITC labeled ghost pen cyclic peptide) selectively binds to F-actin. Ghost pen cyclic peptide derivatives are often used at nanomolar concentrations as convenient probes for labeling, identifying, and quantifying F-actin in formaldehyde fixed and permeabilized tissue sections, cell cultures, or cell-free experiments. The binding of ghost pen cyclic peptide to actin filaments is much tighter than that to actin monomers, resulting in a decrease in the rate constant of actin subunit dissociation from the filament end, thereby stabilizing actin filaments by preventing silk depolymerization. Moreover, it was found that Guibi cyclic peptide inhibits the ATP hydrolysis activity of F-actin. The function of ghost pen cyclic peptide varies at different concentrations in cells. When introduced into the cytoplasm at low concentrations, ghost pen cyclic peptide absorbs cytoplasmic actin and fibrin with lower polymerization degree into the stable "island" of aggregated actin polymer, but it does not interfere with stress fibers, i.e. thick microfilament bundles. The properties of ghost pen cyclic peptide are effective tools for studying the distribution of F-actin in cells. The method is to label ghost pen cyclic peptide with fluorescent analogues and use it to stain actin filaments for optical microscopy observation. The fluorescent derivatives of ghost pen cyclic peptides have been proven to be very useful in locating actin filaments in living or fixed cells, as well as in vitro observation of individual actin filaments. Fluorescent ghost pen cyclic peptide derivatives have been used as important tools for high-resolution study of actin networks.The phalloidin is a toxin isolated from the deadly umbel mushroom. It is a bicyclic peptide that specifically binds to F-actin. Therefore, fluorescent dye-labeled phalloidin can be very convenient to study the distribution of F-actin. Inside the phalloidin, there is an uncommon thioether bridge between cysteine and tryptophan to form an inner ring structure. As pH increases, the thioether is cleaved and the phalloidin loses its affinity for actin.Abs/Em (nm)：546/575 Application scope：Cytoskeleton staining Usage:1. Liquid storage preparationRhodamine labeled ghost pen cyclic peptide: Take an appropriate amount of methanol or sterile water to dissolve the freeze-dried powder in a brown tube, and prepare a 200T/mL storage solution (add 1.5 mL of 300T dye and 0.25 mL of 50T dye).The definition of one unit (T) of Rhodamine labeled ghost pen cyclic peptide is the amount of dye used to stain a loaded cell slide. For Rhodamine labeled ghost pen cyclic peptides, the recommended dilution ratio for use is 1:40-1:200, with one unit equivalent to 200 μ Add 1-5 to the total staining volume of L μ L 200T/mL stock solution.Note: The dilution ratio can be adjusted appropriately based on the actual dyeing effect.2. Fixed cell stainingThe following plan is for the staining steps of adherent cells grown on glass cover slides or 8-well chamber slides. Ghost pen cyclic peptides can also be used for staining fixed frozen or paraffin tissue sections.(1) Wash the cells three times with PBS.(2) Fix cells in PBS solution containing 3.75% formaldehyde and fix on ice for 15 minutes.Note: Methanol can destroy actin during the fixation process. Therefore, it is best to avoid fixatives containing any methanol. The preferred fixative is formaldehyde without methanol.(3) Wash the cells three times with PBS.(4) Permeate cells with PBS solution containing 0.5% Triton X-100 at room temperature for 10 minutes.(5) Wash the cells three times with PBS.(6) Using 200 μ Dilution 1-5 with L PBS μ Add fluorescent labeled ghost pen peptide storage solution to a cover glass slide or well, incubate at room temperature for 20 minutes, and stain.Note: The staining volume can be adjusted according to the sample situation. To avoid the volatilization of the dye solution during the incubation process, the cover glass can be placed in a sealed container.(7) Wash cells 2-3 times with PBS.(8) Fluorescence microscopy observation. The labeled ghost pen cyclic peptide has good photostability, and the sample can be imaged in PBS. However, for optimal results, anti fluorescence quenching agents can also be used for observation.3. Live cell stainingFluorescent labeled ghost pen cyclic peptides do not exhibit cell permeability, therefore they have not been widely used for live cell labeling. However, there are reports that live cells may be labeled through endocytosis or unknown mechanisms. Generally speaking, staining live cells requires more dyes. Alternatively, fluorescent labeled ghost pen cyclic peptides can also be injected into cells to monitor actin distribution and cell movement Points for attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments ; 2.This product is in the form of freeze-dried powder, and the trace is not easy to observe. Before use, please instantaneously centrifuge, add appropriate solvent to dissolve and use, and the solution after dissolution is almost transparent ; 3.If it is prepared into an aqueous solution, it should be stored in small quantities. MDL Number: MFCD00278840 Molecular Formula: C62H72N12O12S4 Molecular Weight: 1305.57 Related Documents: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/P378705-SCI_34676cfd855bd525400fbc714b84a036.pdf