Phalloidin-Tetramethylrhodamine Conjugate

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.

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	~1300
Solvent	DMSO

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 Origin	Download PDF
H-phrase	H301, H311, H331
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R23, R24, R25
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

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

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

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

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

Open in Advanced Spectrum Viewer

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

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

Images

Figure 1. Chemical structure for Phalloidin-Tetramethylrhodamine Conjugate

Figure 2. 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: Tangshenning Attenuates High Glucose-Induced Podocyte Injury via Restoring Autophagy Activity through Inhibiting mTORC1 Activation by Xu et. al., Journal of Diabetes Research June 2022.

Citations

View all 48 citations: Citation Explorer

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

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)

High-manganese and nitrogen stabilized austenitic stainless steel (Fe--18Cr--22Mn--0.65 N): a material with a bright future for orthopedic implant devices
Authors: Kumar, Chandra Shekhar and Singh, Gaurav and Poddar, Suruchi and Varshney, Neelima and Mahto, Sanjeev Kumar and Podder, Arijit Saha and Chattopadhyay, Kausik and Rastogi, Amit and Singh, Vakil and Mahobia, Girija Shankar
Journal: Biomedical Materials (2021): 065011

Targeted core-shell nanoparticles for precise CTCF gene insert in treatment of metastatic breast cancer
Authors: Duan, Jialun and Bao, Chunjie and Xie, Ying and Guo, Haitao and Liu, Yixuan and Li, Jianwei and Liu, Rui and Li, Peishan and Bai, Jing and Yan, Yan and others,
Journal: Bioactive Materials (2021)

DNA biodots based targeted theranostic nanomedicine for the imaging and treatment of non-small cell lung cancer
Authors: Jha, Abhishek and Viswanadh, Matte Kasi and Burande, Ankita Sanjay and Mehata, Abhishesh Kumar and Poddar, Suruchi and Yadav, Kanchan and Mahto, Sanjeev Kumar and Parmar, Avanish Singh and Muthu, Madaswamy S
Journal: International Journal of Biological Macromolecules (2020): 413--425

Simple application of adipose-derived stem cell-derived extracellular vesicles coating enhances cytocompatibility and osteoinductivity of titanium implant
Authors: Chen, Lifeng and Mou, Shan and Hou, Jinfei and Fang, Huimin and Zeng, Yuyang and Sun, Jiaming and Wang, Zhenxing
Journal: Regenerative Biomaterials (2020)

Formulation and in vivo efficacy study of cetuximab decorated targeted bioadhesive nanomedicine for non-small-cell lung cancer therapy
Authors: Viswanadh, Matte Kasi and Vikas, and Jha, Abhishek and Reddy Adena, Sandeep Kumar and Mehata, Abhishesh Kumar and Priya, Vishnu and Neogi, Kaushik and Poddar, Suruchi and Mahto, Sanjeev Kumar and Muthu, Madaswamy S
Journal: Nanomedicine (2020): 2345--2367

Plasmacytoid dendritic cells regulate host immune response to Citrobacter rodentium induced colitis in colon-draining lymph nodes
Authors: P{\"o}ysti, Sakari and Siloj{\"a}rvi, Satu and Toivonen, Raine and H{\"a}nninen, Arno
Journal: European Journal of Immunology (2020)

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