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Phalloidin Conjugates
This orange fluorescent phalloidin conjugate (equivalent to TRITC-labeled phalloidin) selectively binds to F-actins with much higher photostability than the fluorescein-phalloidin conjugates. 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-Tetramethylrhodamine 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 TRITC 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-Tetramethylrhodamine Conjugate working solution
Add 1 µL of Phalloidin-Tetramethylrhodamine 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-Tetramethylrhodamine 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 TRITC filter set.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (280 nm) |
| Phalloidin-AMCA Conjugate | 346 | 434 | 19000 | 0.153 |
| Phalloidin-Fluorescein Conjugate | 498 | 517 | 800001 | 0.35 |
Citations
View all 54 citations: Citation Explorer
Exosomal PGAM1 promotes prostate cancer angiogenesis and metastasis by interacting with ACTG1
Authors: Luo, Jun-qi and Yang, Tao-wei and Wu, Jun and Lai, Hou-hua and Zou, Li-bin and Chen, Wen-bin and Zhou, Xu-min and Lv, Dao-jun and Cen, Sheng-ren and Long, Zi-ning and others,
Journal: Cell Death \& Disease (2023): 502
Authors: Luo, Jun-qi and Yang, Tao-wei and Wu, Jun and Lai, Hou-hua and Zou, Li-bin and Chen, Wen-bin and Zhou, Xu-min and Lv, Dao-jun and Cen, Sheng-ren and Long, Zi-ning and others,
Journal: Cell Death \& Disease (2023): 502
Trackable and highly fluorescent nanocellulose-based printable bio-resins for image-guided tissue regeneration
Authors: Dutta, Sayan Deb and Patil, Tejal V and Ganguly, Keya and Randhawa, Aayushi and Acharya, Rumi and Moniruzzaman, Md and Lim, Ki-Taek
Journal: Carbohydrate Polymers (2023): 121232
Authors: Dutta, Sayan Deb and Patil, Tejal V and Ganguly, Keya and Randhawa, Aayushi and Acharya, Rumi and Moniruzzaman, Md and Lim, Ki-Taek
Journal: Carbohydrate Polymers (2023): 121232
Development of an Asymmetric Hydrophobic/Hydrophilic Ultrathin Graphene Oxide Membrane as Actuator and Conformable Patch for Heart Repair
Authors: Zhang, Xingying and Song, Chen and Nong, Huijia and Xu, Kaige and Wu, Xiaozhuo and Zhong, Wen and Xing, Malcolm and Wang, Leyu
Journal: Advanced Functional Materials (2023): 2300866
Authors: Zhang, Xingying and Song, Chen and Nong, Huijia and Xu, Kaige and Wu, Xiaozhuo and Zhong, Wen and Xing, Malcolm and Wang, Leyu
Journal: Advanced Functional Materials (2023): 2300866
One-step preparation of novel Ti--Cl bifunctional microstructured surfaces with excellent stem cell compatibility and antibacterial activity
Authors: Shi, Kaihui and Zhang, Hao and Ye, Zhiqian and Liu, Jiangwen and Xie, Guie
Journal: Ceramics International (2023)
Authors: Shi, Kaihui and Zhang, Hao and Ye, Zhiqian and Liu, Jiangwen and Xie, Guie
Journal: Ceramics International (2023)
Qingda granule ameliorates vascular remodeling and phenotypic transformation of adventitial fibroblasts via suppressing the TGF-$\beta$1/Smad2/3 pathway
Authors: Wu, Meizhu and Zhang, Siyu and Zhang, Wenqiang and Zhou, Yuting and Guo, Zhi and Fang, Yi and Yang, Yanyan and Shen, Zhiqing and Lian, Dawei and Shen, Aling and others,
Journal: Journal of Ethnopharmacology (2023): 116535
Authors: Wu, Meizhu and Zhang, Siyu and Zhang, Wenqiang and Zhou, Yuting and Guo, Zhi and Fang, Yi and Yang, Yanyan and Shen, Zhiqing and Lian, Dawei and Shen, Aling and others,
Journal: Journal of Ethnopharmacology (2023): 116535
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
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
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
Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
Authors: Vig A, Dudas R, Kupi T, Orban J, Hild G, Lorinczy D, Nyitrai M.
Journal: J Therm Anal Calorim (2009): 721
Authors: Vig A, Dudas R, Kupi T, Orban J, Hild G, Lorinczy D, Nyitrai M.
Journal: J Therm Anal Calorim (2009): 721
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