Phalloidin-Cell Skeleton Staining Wizard

Phalloidin-Cell Skeleton Staining Wizard

The cell skeleton is a reticular fiber structure that is highly dynamic and exhibits different forms at different stages. It is mainly composed of microfilaments, microtubules, and intermediate filaments. Microfilaments are primarily composed of actin, which exists in two forms: monomeric globular actin (G-actin) and polymeric filamentous actin (F-actin). The assembly of microfilaments involves the conversion of G-actin into F-actin.

Phalloidin is a type of bicyclic heptapeptide, one of the earliest cyclic peptides discovered, isolated from the death cap mushroom (Amanita phalloides). It functions by binding to and stabilizing filamentous actin (F-actin) and effectively preventing the depolymerization of actin fibers. Due to its tight and selective binding to F-actin, it reveals the distribution of the microfilament skeleton in cells. Therefore, fluorescence labeling containing Phalloidin has been widely used in biological microscopy techniques.

Arcegen Biological offers a variety of Phalloidin products, mainly distinguished by different fluorescent groups used for labeling, resulting in different fluorescent colors upon excitation. This allows for the selection of appropriate machinery to meet the requirements of excitation and emission wavelengths, thereby distinguishing the fluorescent colors of other fluorescent dyes during co-staining.

 

Experimental Procedure

Preparation of Phalloidin

Dissolve 0.1mg of Phalloidin in 1ml of anhydrous methanol (can also be dissolved in DMSO or anhydrous ethanol) to prepare the stock solution, which can be stored long-term at -20°C in the dark. The working solution concentration is 5ug/ml, diluted 20 times with PBS from the stock solution.

Staining Procedure

Wash cells twice with PBS for 10 minutes each time. Fix cells with 3.7%-4% formaldehyde or paraformaldehyde for 20 minutes, then wash cells twice with PBS. Add 5ug/ml FITC-Phalloidin for room temperature staining for 30-60 minutes (shorter time in summer). Wash cells twice with PBS. Stain cell nuclei with DAPI or Hoechst33258 for 10 minutes, then wash cells twice with PBS. Remove excess moisture, add fluorescence mounting medium (neutral or slightly alkaline buffer with equal parts glycerol), cover with a coverslip, and observe under a fluorescence or confocal microscope.

Customer Examples

Customer Source: Sichuan University West China Hospital, School of Stomatology

Cell Type: Bone Marrow Mesenchymal Stem Cells

Scale: 100μm

Reference: Zhu Z, Jiang S, Liu Y, et al. Micro or nano: Evaluation of biosafety and biopotency of magnesium metal organic framework-74 with different particle sizes. Nano Research, 2020, 13(2):511-526.

Product Introduction: The product used for staining is FITC-labeled Phalloidin, which exhibits green fluorescence. The image was obtained using MTT assay and FITC-labeled Phalloidin and DAPI staining to determine the safe dosage of Mg-MOF74 for BMSCs, ensuring subsequent experiments are conducted within the safe dosage range.

Cell Type: Bone Marrow Mesenchymal Stem Cells

Scale: 50μm

Reference: Fan Z, Xiao L, Lu G, Ding Z, Lu Q. Water-insoluble amorphous silk fibroin scaffolds from aqueous solutions. J Biomed Mater Res B Appl Biomater. 2020;108(3):798-808. doi:10.1002/jbm.b.34434

Product Introduction: The product used for staining is Rhodamine-labeled Phalloidin, which exhibits red fluorescence. The image shows Rhodamine-labeled Phalloidin staining and DAPI nuclear staining, observing the differentiation of bone marrow mesenchymal stem cells into endothelial cells on different regenerated silk fibroin scaffolds under confocal microscopy.

 

Conclusion

Fluorescent and Biotinylated Derivatives of Phalloidin Peptides offer significant advantages over actin antibodies. The binding of Phalloidin peptide conjugates remains consistent across species (including animals and plants) and is not significantly affected by variations in actin. Furthermore, their nonspecific staining is negligible, resulting in high contrast between stained and unstained regions.

 

Considerations

1. Phalloidin peptides have low molecular weight and easily penetrate cells without the need for pre-treatment with cold acetone or TRITON X-100.

2. They exhibit high toxicity; therefore, wear gloves while handling.

 

Product Features

(1) High affinity: Kd= 20 nM;

(2) Strong specificity: selectively binds to filamentous actin (F-actin) without binding to monomeric globular actin (G-actin);

(3) Superior to antibody staining: Phalloidin peptides are not species-restricted and exhibit minimal nonspecific staining, resulting in extremely clear contrast between stained and unstained regions;

(4) High sensitivity: staining at nanomolar concentrations (nM) meets experimental requirements;

(5) Good compatibility and actin activity preservation: Phalloidin peptide derivatives are small, with a diameter of approximately 12-15Å and a molecular weight <2000 Da, allowing many physiological properties of labeled actin to be maintained;

(6) Wide applicability: No species specificity; equally suitable for formaldehyde-fixed and perforated tissue samples.

 

 FAQ
Q1: Can Phalloidin peptide staining be performed in live cells?

A: Phalloidin peptide staining requires sample fixation and permeabilization to facilitate binding between Phalloidin peptides and F-actin, resulting in better staining results.

Q2: My cell samples have been transfected with a GFP-labeled plasmid. Which Phalloidin peptide should I choose?

A: You can choose Phalloidin peptides labeled with TRITC (orange), iFluorTM 555 (orange), or iFluorTM 647 (far-red).

Q3: How much working solution is needed for staining adherent cells?

A: Staining adherent cells only requires enough staining working solution to completely cover the cells.

Q4: What are the differences between different Phalloidin peptides, and how should I choose?

A: The differences lie in the fluorescent groups used for labeling, resulting in different fluorescent colors upon excitation. This helps distinguish the fluorescent colors of other fluorescent dyes during co-staining. Choose based on the machine's requirements for excitation and emission wavelengths.

Q5: Is there species specificity in Phalloidin peptide staining?

A: There is no species specificity for staining live animal cells.

 

Related Products

Product Name    

Catalog Number

Specifications

Phalloidin

C331201E

1 mg

Phalloidin

C331201S

5 mg

Amino-Phalloidin

C331202E

1 mg

TRITC Phalloidin 

C331207E

300 T

TRITC Phalloidin 

C331207S

1 mg

FITC Phalloidin 

C331203E

300 T

FITC Phalloidin 

C331203S

1 mg

Fluor 488 phalloidin, Green

C331204E

300 T

Fluor 555 phalloidin, Orange

C331205E

300 T

Fluor 647 phalloidin, Far Infrared

C331206E

300 T


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