Principle of ghost pen peptide staining (Alexa Fluor 488 labeling ghost pen peptide usage method)
Ghost pen cyclic peptide is a bicyclic heptapeptide toxin that selectively binds to fibrous actin (F-actin) in animals and plants, making it a powerful tool for studying intracellular actin microfilaments (F-actin). Under an optical microscope, fluorescently labeled phalloidin dye conjugate can clearly display the morphology and distribution of intracellular microfilaments, and is widely used in biomedical research.
Principle of ghost pen cyclic peptide staining
Fluorescent labeled ghost pen peptides can selectively bind to F-actin at nanogram levels and are easily soluble in water, providing a very convenient labeling method for detecting actin localization and quantification in tissue sections, cultured cells, and cell-free systems. Through its fluorescence properties, it emits fluorescence under specific wavelength light excitation, making the structure and distribution of actin filaments in cells clearly visible.
Usage of Ghost Pen Cyclic Peptide
Taking Alexa Fluor 488 as an example to mark ghost pen cyclic peptide (green)
Step 1: Prepare 1 x Alexa Fluor 488 labeled ghost pen peptide working solution. Transfer 1 μ L of 1000 x Alexa Fluor 488 labeled ghost pen peptide storage solution to 1 mL of PBS buffer containing 1% BSA to obtain 1 x working solution.
Step 2: Cell staining procedure
(1) Cell culture overnight or longer to achieve a density of 50-60% confluence.
(2) Remove the culture medium and wash the cells twice with 1 x PBS (pH 7.4) preheated at 37 ℃.
(3) Use PBS solution containing 3-4% formaldehyde for cell fixation, and fix at room temperature for 10-30 minutes.
(4) Wash the fixed cells with PBS 2-3 times at room temperature for 10 minutes each time.
(5) (Optional): At room temperature, permeabilize with 0.1% Triton X-100 solution dissolved in PBS for 3-5 minutes to increase its permeability.
(6) Wash cells 2-3 times with PBS at room temperature for 10 minutes each time.
(7) Add 100 μ L/well (96 well plate) of Alexa Fluor 488 labeled ghost pen cyclic peptide working solution to each well, and stain at room temperature in the dark for 20-90 minutes, with relatively shorter time in summer.
(8) Wash the cells three times with PBS, each time for 5 minutes, to remove excess Alexa Fluor 488 labeled ghost pen peptides.
(9) (Optional): Add sufficient ready to use DAPI solution for counterstaining of cell nuclei, such as 100 μ L/well (96 well plate), at room temperature for 3-5 minutes. Wash the cells twice with PBS for 5 minutes each time.
(10) Perform fluorescence observation under a fluorescence microscope or confocal microscope, using Alexa FluorTM 488 excitation/emission filter and/or DAPI excitation/emission filter (Ex/Im=364/454nm).
Precautions for Ghost Pen Cyclic Peptide
✔ Storage conditions: Ghost pen cyclic peptide and its fluorescently labeled derivatives should be stored at -20 ℃ or below, kept dry and away from light.
✔ Cell processing: Before performing ghost pen peptide staining, cells should undergo appropriate fixation and permeation treatment to ensure staining effectiveness.
✔ Fluorescence microscope setup: Adjust the fluorescence microscope setup according to the excitation and emission wavelengths of the fluorescent labeled phalloidin used to achieve the best observation effect.
✔ The staining situation of different cells varies, and the corresponding usage of Alexa Fluor ghost pen peptide also needs to be determined according to different situations.
✔ Highly toxic, gloves are required for operation
✔ Avoid the presence of methanol in fixatives, as methanol may damage actin during the fixation process.
488 Green Ghost Pen Peptide Product Recommendation
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