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Phalloidin is a cyclic heptapeptidetic toxin derived from Amanita phalloides. It selectively binds to filamentous actin F-actin with high affinity (Kd= 20 nM), but does not bind to monomeric actin G-actin. It is commonly used to label F-actin in tissue sections, cell cultures, or cell-free systems for qualitative and quantitative analysis of F-actin. In addition, porcinocyclic peptide derivatives also bind with similar affinity to large and small fibers, whether muscle cells or non-muscle cells of animal or plant origin, in accordance with the stoichimetric ratio of about one porcinocyclic peptide molecule per actin subunit. The nonspecific binding was almost negligible, and the identification of stained and non-stained regions was very obvious. Therefore, porcinocyclic peptide derivatives are particularly suitable to replace Actin antibodies for related research. In addition, the derivatives are small, about 12-15A in diameter and < 2000 Daltons in molecular weight. Many physiological properties of unlabeled Actin can be maintained, for example, actin binding proteins such as myosin, tropomyosin and DNase I can still react. The filaments labeled with phlebocyclin could still penetrate the solid myosin matrix. And the muscle fibers extracted by glycerol can still shrink after labeling.
Binding of Phalloidin prevents the dissociation of filamentous actin (microfilaments), stabilizes the microfilament structure, and thus disrupts the polymerization-depolymerizational homeostasis of microfilaments. This property reduces the critical concentration (CC) at which actin polymerization occurs to < 1μg/mL and, therefore, can be used as a polymerization accelerator. In addition, porcinocyclic peptide can also inhibit the ATP hydrolysis activity of F-actin.This product is TRITC (tetramethylRhodamine isothiocyanate) labeled ghost ring peptide, strong staining reaction specificity, high contrast, has better staining effect than Actin antibody, suitable for qualitative and quantitative detection of F-actin. In addition, F-actin combined with this product can still maintain many biological properties of actin itself. The combination of this product has no species difference and has wide applicability.
1. Preparation of working fluid
This product is provided in the form of a 20μM storage solution dissolved in methanol for a total of 300μl. According to the concentration of 100 nM working liquid, the total amount of 60 ml working liquid can be prepared. It is recommended that after receiving the product, according to the single amount of use, the mother liquor is packaged in a small amount, frozen at -20℃ away from light, and stable for one year. Before starting the test, dilute the storage solution to the required working concentration using 1×PBS buffer. Recommended working concentration: 80~200nM. Use it now.
2. Dyeing steps
1) Cells were grown for 24h, and the density reached 50% confluent degree.
2) The culture medium was sucked off, and the cells were cleaned twice with 1×PBS (pH 7.4) preheated at 37℃.
3) Cell fixation was performed with 4% formaldehyde solution dissolved in PBS at room temperature for 10min. Note: Avoid the fixative containing methanol, as methanol may destroy actin during the fixative process.
4) At room temperature, the cells were cleaned with PBS for 2 to 3 times, 10min each time.
5) At room temperature, dehydrated with acetone (≤-20℃) or permeated with 0.5% Triton X-100 solution for 5min.
6) At room temperature, the cells were cleaned with PBS for 2 to 3 times, 10min each time.
7) 200μl of TRITC-labeled ghost pen cyclic peptide working liquid was taken, the cells on the cover glass were covered, and incubated at room temperature for 30min away from light (incubation at 4℃~37℃ is generally OK). Note: In order to reduce the background, 1% BSA can be added to the TRITC-labeled ghost cyclic peptide working solution; In addition, to avoid solution flapping during incubation, the cover glass can be transferred to an airtight container.
8) Clean the cover glass with PBS for 3 times, 5min each time.
9) Restain the nucleus with 200μl DAPI solution (concentration: 100 nM) for about 30s.
10) Wash the cover glass with PBS, then invert it on a slide already coated with a drop of Fluoromount-GTM water-soluble sealer. Gently wipe off excess sealer with a paper towel, then seal the sealer permanently with nail polish. The specimen slides prepared by this method can be stored at 4 ° C away from light and can usually be continued for F-actin staining analysis within 6 months. Note: It is also possible to simplify the procedure by directly using an anti-fluorescence quenched tablet containing DAPI combined with steps 9) 10).
11) Fluorescence observation was performed under fluorescence microscope or confocal microscope. TRITC excitation/emission filter (Ex/Em=540/570nm) and DAPI excitation/emission filter (Ex/Em=364/454nm) were selected.
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
