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Phalloidin Conjugates
This blue fluorescent phalloidin conjugate (equivalent to coumarin-labeled phalloidin) selectively binds to F-actins. Used at nanomolar concentrations, phalloidin derivatives are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. Phalloidin binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the rate constant for the dissociation of actin subunits from filament ends, essentially stabilizing actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin. Phalloidin functions differently at various concentrations in cells. When introduced into the cytoplasm at low concentrations, phalloidin recruits the less polymerized forms of cytoplasmic actin as well as filamin into stable "islands" of aggregated actin polymers, yet it does not interfere with stress fibers, i.e. thick bundles of microfilaments. The property of phalloidin is a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain actin filaments for light microscopy. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in living or fixed cells as well as for visualizing individual actin filaments in vitro. Fluorescent phalloidin derivatives have been used as an important tool in the study of actin networks at high resolution. AAT Bioquest offers a variety of fluorescent phalloidin derivatives with different colors for multicolor imaging applications.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare samples in microplate wells
- Remove liquid from samples in the plate
- Add Phalloidin-AMCA Conjugate solution (100 μL/well)
- Stain the cells at room temperature for 20 to 90 minutes
- Wash the cells
- Examine the specimen under microscope with DAPI filter
Storage and Handling Conditions
The solution should be stable for at least 6 months if store at -20 °C. Protect the fluorescent conjugates from light, and avoid freeze/thaw cycles.Note Phalloidin is toxic, although the amount of toxin present in a vial could be lethal only to a mosquito (LD50 of phalloidin = 2 mg/kg), it should be handled with care.
PREPARATION OF WORKING SOLUTION
Phalloidin-AMCA Conjugate working solution
Add 1 µL of Phalloidin-AMCA Conjugate solution to 1 mL of PBS with 1% BSA.Note The stock solution of phalloidin conjugate should be aliquoted and stored at -20 °C. protected from light.
Note Different cell types might be stained differently. The concentration of phalloidin conjugate working solution should be prepared accordingly.
SAMPLE EXPERIMENTAL PROTOCOL
Stain the cells
- Perform formaldehyde fixation. Incubate cells with 3.0–4.0 % formaldehyde in PBS at room temperature for 10–30 minutes.
Note Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde. - Rinse the fixed cells 2–3 times in PBS.
- Optional: Add 0.1% Triton X-100 in PBS into fixed cells for 3 to 5 minutes to increase permeability. Rinse the cells 2–3 times in PBS.
- Add 100 μL/well (96-well plate) of Phalloidin-AMCA Conjugate working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.
- Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope with DAPI filter set.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (260 nm) | Correction Factor (280 nm) |
| Phalloidin-Fluorescein Conjugate | 498 | 517 | 800001 | 0.32 | 0.35 |
| Phalloidin-Tetramethylrhodamine Conjugate | 552 | 578 | 90000 | 0.32 | 0.178 |
Citations
View all 28 citations: Citation Explorer
Tissue-type plasminogen activator regulates p35-mediated Cdk5 activation in the postsynaptic terminal
Authors: Diaz, Ariel and Jeanneret, Valerie and Merino, Paola and McCann, Patrick and Yepes, Manuel
Journal: Journal of cell science (2019): jcs224196
Authors: Diaz, Ariel and Jeanneret, Valerie and Merino, Paola and McCann, Patrick and Yepes, Manuel
Journal: Journal of cell science (2019): jcs224196
Biomaterial Surface Can Modify HUVEC Morphology and Inflammatory Response by Regulating MicroRNA Expression
Authors: Gu, Shuangying and Tian, Baoxiang and Chen, Weicong and Zhou, Yue
Journal: Journal of Biosciences and Medicines (2017): 8
Authors: Gu, Shuangying and Tian, Baoxiang and Chen, Weicong and Zhou, Yue
Journal: Journal of Biosciences and Medicines (2017): 8
DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes
Authors: Sun, Ming-Hong and Yang, Mo and Xie, Feng-Yun and Wang, Wei and Zhang, Lili and Shen, Wei and Yin, Shen and Ma, Jun-Yu
Journal: PloS one (2017): e0170308
Authors: Sun, Ming-Hong and Yang, Mo and Xie, Feng-Yun and Wang, Wei and Zhang, Lili and Shen, Wei and Yin, Shen and Ma, Jun-Yu
Journal: PloS one (2017): e0170308
Grafting of Ring-Opened Cyclopropylamine thin films on Silicon (100) Hydride via UV Photoionization
Authors: Tung, Joline and Ching, Jing Yuan and Ng, Yoke Mooi and Tew, Lih Shin and Khung, Yit Lung
Journal: ACS Applied Materials & Interfaces (2017)
Authors: Tung, Joline and Ching, Jing Yuan and Ng, Yoke Mooi and Tew, Lih Shin and Khung, Yit Lung
Journal: ACS Applied Materials & Interfaces (2017)
Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery
Authors: Chang, Ting-Ya and Chen, Chen and Lee, Min and Chang, Ya-Chu and Lu, Chi-Huan and Lu, Shao-Tzu and Wang, De-Yao and Wang, Aijun and Guo, Chin-Lin and Cheng, Pei-Lin
Journal: eLife (2017): e31101
Authors: Chang, Ting-Ya and Chen, Chen and Lee, Min and Chang, Ya-Chu and Lu, Chi-Huan and Lu, Shao-Tzu and Wang, De-Yao and Wang, Aijun and Guo, Chin-Lin and Cheng, Pei-Lin
Journal: eLife (2017): e31101
References
View all 127 references: Citation Explorer
Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes
Authors: Lin G, Qiu X, F and el TM, Albersen M, Wang Z, Lue TF, Lin CS.
Journal: Urology (2011): 970 e1
Authors: Lin G, Qiu X, F and el TM, Albersen M, Wang Z, Lue TF, Lin CS.
Journal: Urology (2011): 970 e1
Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin
Authors: Diensthuber RP, Muller M, Heissler SM, Taft MH, Chizhov I, Manstein DJ.
Journal: FEBS Lett (2011): 767
Authors: Diensthuber RP, Muller M, Heissler SM, Taft MH, Chizhov I, Manstein DJ.
Journal: FEBS Lett (2011): 767
Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation
Authors: An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM.
Journal: Proc Natl Acad Sci U S A (2010): 20246
Authors: An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM.
Journal: Proc Natl Acad Sci U S A (2010): 20246
Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity
Authors: Herraez E, Macias RI, Vazquez-Tato J, Hierro C, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 21
Authors: Herraez E, Macias RI, Vazquez-Tato J, Hierro C, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 21
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