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Phalloidin-Tetramethylrhodamine Conjugate

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Effects of TSN on nephrin expression and podocyte cytoskeleton in high glucose-induced podocytes. Staining of podocytes cytoskeleton with phalloidin-rhodamine, original magnification ×630, scale 50 μm. Source: <b>Tangshenning Attenuates High Glucose-Induced Podocyte Injury via Restoring Autophagy Activity through Inhibiting mTORC1 Activation</b> by Xu <em>et. al.</em>, <em>Journal of Diabetes Research</em> June 2022.
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Physical properties
Molecular weight~1300
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.178
Extinction coefficient (cm -1 M -1)90000
Excitation (nm)552
Emission (nm)578
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH301, H311, H331
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR23, R24, R25
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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Show More (47)

OverviewpdfSDSpdfProtocol


Molecular weight
~1300
Correction Factor (260 nm)
0.32
Correction Factor (280 nm)
0.178
Extinction coefficient (cm -1 M -1)
90000
Excitation (nm)
552
Emission (nm)
578
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
  1. Prepare samples in microplate wells
  2. Remove liquid from samples in the plate
  3. Add Phalloidin-Tetramethylrhodamine Conjugate solution (100 μL/well)
  4. Stain the cells at room temperature for 20 to 90 minutes
  5. Wash the cells
  6. Examine the specimen under microscope with TRITC filter 
Important      Warm the vial to room temperature and centrifuge briefly before opening.

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
  1. 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.
  2. Rinse the fixed cells 2–3 times in PBS.
  3. 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.
  4. 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.
  5. 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


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spectrum

Spectral properties

Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.178
Extinction coefficient (cm -1 M -1)90000
Excitation (nm)552
Emission (nm)578

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (280 nm)
Phalloidin-AMCA Conjugate346434190000.153
Phalloidin-Fluorescein Conjugate4985178000010.35

Images


Citations


View all 54 citations: Citation Explorer
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Authors: Shi, Kaihui and Zhang, Hao and Ye, Zhiqian and Liu, Jiangwen and Xie, Guie
Journal: Ceramics International (2023)
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
Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase
Authors: Li, Lianxin and Xin, Jinge and Wang, Hesong and Wang, Yadong and Peng, Weiqi and Sun, Ning and Huang, Haonan and Zhou, Yanxi and Liu, Xingmei and Lin, Yu and others,
Journal: Ecotoxicology and Environmental Safety (2023): 114940
Methyltransferase KDKE motif influences the intercellular transmission of Newcastle disease virus
Authors: Li, Xiao and Zhao, Ye and Teng, Qing-Yuan and Zhang, Xue-Hui and Xue, Jia and Zhang, Guo-Zhong
Journal: Virulence (2023): 2186336
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Journal: Carbohydrate Polymers (2023): 121232
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
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
Tangshenning Attenuates High Glucose-Induced Podocyte Injury via Restoring Autophagy Activity through Inhibiting mTORC1 Activation
Authors: Xu, Jiayi and Shan, Xiaomeng and Chen, Chunwei and Gao, Yanbin and Zou, Dawei and Wang, Xiaolei and Wang, Tao and Shi, Yimin
Journal: Journal of Diabetes Research (2022)
Hepatocyte-targeting and tumor microenvironment-responsive liposomes for enhanced anti-hepatocarcinoma efficacy
Authors: Cheng, Dongliang and Wen, Zhiwei and Chen, Hui and Lin, Shiyuan and Zhang, Wei and Tang, Xin and Wu, Wei
Journal: Drug Delivery (2022): 2995--3008
Melatonin prevents oocyte deterioration due to cotinine exposure in mice
Authors: Cheng, Jinmei and Mi, Panpan and Li, Yinchuan and Lu, Yajuan and Sun, Fei
Journal: Biology of Reproduction (2022)

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
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
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
Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
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
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
In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives
Authors: Herraez E, Macias RI, Vazquez-Tato J, Vicens M, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 13
Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes
Authors: Luo H, Hallen-Adams HE, Walton JD.
Journal: J Biol Chem (2009): 18070
Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations
Authors: Wollesen T, Loesel R, Wanninger A.
Journal: J Neurosci Methods (2009): 63
Anti-acetylated tubulin antibody staining and phalloidin staining in the starlet sea anemone Nematostella vectensis
Authors: Genikhovich G, Technau U.
Journal: Cold Spring Harb Protoc (2009): pdb prot5283