Phalloidin-TFAX 488 |
Catalog No.GC50523 |
Green fluorescent cytoskeletal stain; binds F-actin
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 289620-19-5
Sample solution is provided at 25 µL, 10mM.
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Product Documents
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Protocol
This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare dyeing solution
(1) Dye stock solution: Take the lyophilized powder stored at low temperature and return it to room temperature for at least 20 minutes. After centrifugation at low speed, add methanol or anhydrous DMSO to fully dissolve it to prepare a 10-100 μM stock solution. The prepared stock solution should be aliquoted and stored in the dark at -20 or -80°C;
(2) Dye working solution: It is recommended to use PBS (the PBS used in this solution is 1xPBS with pH 7.4) to dilute the stock solution and prepare a working solution with a concentration of 10-200nM.
Notice:
① Please adjust and optimize the working fluid concentration according to the actual situation, and prepare it now.
② A PBS dilution stock solution containing 1% BSA can be used to reduce non-specific background staining and minimize the possibility of phalloidin adhering to the tube wall.
2. Cell staining
(1) Cells are cultured on slides and grown to reach 70-80% confluence.
(2) Aspirate the culture medium and wash the cells twice with 37°C preheated PBS.
(3) Fix the cells with 4% paraformaldehyde dissolved in PBS and fix at room temperature for 10 minutes.
Notice:
① Methanol can destroy actin during the fixation process. It is recommended to use methanol-free fixative;
② Cells can also be fixed in PBS containing 3-4% formalin at room temperature for 10-30 minutes.
(4) Wash cells 2-3 times with PBS at room temperature, 30 seconds each time.
Optional step ①: Treat cells with PBS containing 10 mM ethanolamine (or 0.1 M glycine) for 5 minutes to quench excess formalin;
Optional step ②: Add PBS containing 0.1% Triton X-100 to the fixed cells, let stand for 3-5 minutes to increase permeability, and then wash the cells 2-3 times with PBS.
(5) Take about 100 μl of the freshly prepared staining working solution to completely cover the cells on the coverslip, and incubate at room temperature in the dark for 30 minutes.
Notice:
① Generally, 4℃-37℃ is suitable for dyeing. In order to avoid evaporation of the working solution, place the coverslip in a sealed container during the incubation process;
② If necessary, a nuclear staining solution with a different fluorescence spectrum than TFAX 488 can be added at this time.
(6) Wash cells 2-3 times with PBS at room temperature, 30 seconds each time.
(7) Use filter paper to absorb as much liquid as possible on the cell surface, place the coverslip upside down on a glass slide with a drop of anti-fluorescence quenching agent, gently absorb the excess quenching agent with a paper towel, and then seal the coverslip with transparent nail polish All around. F-actin staining can still be maintained if the slides treated by this method are stored at 4°C in the dark for at least 6 months.
(8) Observe the staining results under a fluorescence microscope. The maximum excitation/emission light of Phalloidin-TFAX 488 is 490/520nm.
Precautions:
① Cells can be stained in cell culture plates/copolymer dishes. Step (7) is changed to adding anti-fluorescence quenching agent dropwise into the wells to protect fluorescence;
② Phalloidin staining is not suitable for cells fixed with methanol or acetone. Such fixatives will destroy the structure of actin and prevent phalloidin from staining. It is recommended to use 0.2% glutaraldehyde to fix cells;
③ Phalloidin is sensitive to pH: If the pH value increases, the key thioether bridge in phalloidin will be cleaved, thereby losing its affinity for actin;
④ Phalloidin staining can be used in conjunction with antibody staining. It is recommended to add phalloidin conjugate during the incubation with primary or secondary antibodies;
⑤ The optimal concentration and incubation time of phalloidin conjugate depends on the specific cell type, fixation/sample preparation conditions, and/or cell/tissue permeability to the probe;
⑥ Suspension cells can be attached to poly-D-lysine microplates or coverslips, and then stained using the adherent cell protocol;
⑦ If the cell condition is poor, it is recommended to add serum (2-10%) to the staining solution and washing solution;
⑧ In some cases, a one-step method can be used for rapid phalloidin staining: 3.7% formalin and 50-100µg/mL palmitoyl lysophosphatidylcholine coupled with phalloidin at 4°C. Incubate in the conjugate for 20 minutes, then wash 3 times and mount;
⑨ When staining in unfixed samples, phalloidin binding reduces the rate of dissociation of actin subunits from actin filament ends, thereby stabilizing actin filaments by preventing actin filament depolymerization. ;
⑩ For the staining process of other sample types, in order to optimize the fixation conditions and facilitate staining, it is recommended to change the fixation time and formalin concentration within a certain range;
⑪ Phalloidin can be used for formalin-fixed and permeabilized tissue sections, cell cultures and other sample types as well as cell-free experiments. It can also be used for dewaxed paraffin-embedded samples, and phalloidin staining is not possible. Antigen retrieval is required;
⑫ The LD50 of phalloidin is 2 mg/kg. Please pay attention to protection when using it. But usually, phalloidin is used in very small amounts and does not pose a significant safety risk;
⑬ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;
For your safety and health, please wear a lab coat and disposable gloves.
Green fluorescent cytoskeletal stain. Composed of the F-actin probe, Phalloidin , conjugated to TFAX 488. Suitable for use in super resolution microscopy techniques such as dSTORM. Excitation/emission maximum λ ~ 495/418 nm. Water soluble. We suggest making up a stock solution of this product by dissolving the vial contents in 1.5 mL methanol or DMSO. Please refer to the protocol tab for further information on using this product.
Panchuk-Voloshina et al (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J.Histochem.Cytochem. 47 1179 PMID:10449539
Cas No. | 289620-19-5 | SDF | |
Canonical SMILES | O=C([C@H](N1)CSC(NC2=C3C=CC=C2)=C3C[C@@H](C(N[C@@H](C[C@](O)(C)CNC(C4=CC(C(O[Li])=O)=C(C5=C(C=C6)C(OC7=C5C=CC(N)=C7S(=O)(O[Li])=O)=C(S(=O)([O-])=O)C6=[NH2+])C=C4)=O)C(NC(C(N[C@H]([C@H](C)O)C1=O)=O)C)=O)=O)NC([C@@H](NC8=O)C)=O)N9[C@H]8C[C@@H](O)C9 | ||
Formula | C56H59Li2N11O20S3 | M.Wt | 1316.21 |
Solubility | Soluble in DMSO | Storage | Store at -20°C |
General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
||
Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. |
Complete Stock Solution Preparation Table
Prepare stock solution | |||
1 mg | 5 mg | 10 mg | |
1 mM | 0.7598 mL | 3.7988 mL | 7.5976 mL |
5 mM | 0.152 mL | 0.7598 mL | 1.5195 mL |
10 mM | 0.076 mL | 0.3799 mL | 0.7598 mL |
In vivo Formulation Calculator (Clear solution)
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Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )
Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
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