Acti-stain 555 phalloidin

Acti-stain 555 phalloidin
$0.00

Product Uses Include

    • Fluorescent staining of actin filaments in fixed tissue sections and tissue culture cells preparations.Note: Unlike many actin antibodies, Acti-stain™ 555 phalloidin binds only to F-actin resulting in low background fluorescence.  Furthermore, actin staining is not appreciably different between species.
    • Preparation of stabilized fluorescent actin filaments in vitro.

       

      Actin staining is very useful in determining the structure and function of the cytoskeleton in living a nd fixed cells. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed with paraformaldehyde prior to probing or staining for actin structures. 

       

      Material

      Phalloidin is a seven amino acid peptide toxin from the mushroom Amanita phalloides, which binds specifically and with high affinity (Kd 20 nM) to the polymerized form of actin (F-actin).  Phalloidin lowers the critical concentration of actin polymerization to less than 1 µg/ml, thereby acting as a polymerization enhancer. Phalloidin has been labeled with a proprietary red fluorescent dye which allows it to be used to stain actin filaments in tissue cultured cells and tissue sections (1, see Figure 1) and cell-free preparations.  Acti-stain™ 555 phalloidin-labeled actin filaments retain many functional characteristics of unlabeled actin including their ability to interact with myosin.  Actin-stain™ 555 phalloidin is supplied as a red solid. 

      Note: Phalloidin is toxic and must be handled with care (LD50 human = 2mg/Kg).

       


       

      Example Results and Specifications

      Figure 1.   Actin Stress Fibers stained with Acti-stain™ 555 in a Swiss 3T3 cell.

      PHDH1_img1

      Swiss 3T3 cell with activated Rho, nucleus is stained with Dapi (blue) and F-actin is stained with Acti-stain™ 555 (red F-actin, Cat.# PHDH1).

      Figure 2. Emission and excitation scans for Acti-stain™ phalloidins

      PHDH1_scan

      Absorbance and fluorescence scan of Acti-stain™ 555. Labeled phalloidin was diluted into methanol and its absorbance and excitation spectra were scanned between 300-750 and 560-750 nm, respectively. Absorbance peaks at 560 nm and fluorescence at 575 nm.

      Acti-stain phalloidins are the most well characterized phalloidins available. Tabe 1 describes their brightness, photostability, background and affinity constants to F-actin. Compare these performance characteristics to other fluorescent phalloidins and you will see the advantages of using Acti-stain™ for your actin staining requirements.

      Table 1. Biochemical characteristics of fluorescent phalloidins

      ConjugateCat.#

      Wavelengths (Ex/Em)

      Brightness (AFU*)

      Stability to photobleaching(half life in seconds**)

      Background (% of total AFU at 100nM**)

      Affinity (Kd in nanomolar)

      Fluorescein-phalloidin

      na485/535 FITC filter set43262272 +/-12

      Acti-stain™ 488

      PHDG1485/535 FITC filter set83257555 +/-8

      Acti-stain™ 535

      PHDR1535/585 TRITC filter set430271236 +/-7

      Acti-stain™ 555

      PHDH1535/585 TRITC filter set551461663 +/-8

      Acti-stain™ 670

      PHDN1640/680 Cy5 filter set33281850 +/-12

      * = AFU's measured by quantitative cell imaging. ** = Measured in stained Swiss 3T3 cells in the absence of antifade.

       

      For more information about protocols to fix and stain cells click here.

       

      References

      1. Wulf, E. et al. (1979). Proc Natl Acad Sci USA. 76(9):4498-4502.

      2. Kron, S.J. et al. (1991).  Meth. Enzmol. 196: 399-416.

      For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

      Bokelmann, Marcel et al. “Utility of primary cells to examine NPC1 receptor expression in Mops condylurus, a potential Ebola virus reservoir.” PLoS neglected tropical diseases vol. 14,1 e0007952. 21 Jan. 2020, doi:10.1371/journal.pntd.0007952

      Liu, Yi et al. “Effect of F-actin and Microtubules on Cellular Mechanical Behavior Studied Using Atomic Force Microscope and an Image Recognition-Based Cytoskeleton Quantification Approach.” International journal of molecular sciences vol. 21,2 392. 8 Jan. 2020

      Chen, L., Choi, C. S. W., Sanchez-Arias, J. C., Arbour, L. T. & Swayne, L. A. Ankyrin-B p.S646F undergoes increased proteasome degradation and reduces cell viability in the H9c2 rat ventricular cardiomyoblast cell line. Biochem. Cell Biol. 98, 299–306 (2020).

      Chemla, Y. et al. Carbon nanostructures as a scaffold for human embryonic stem cell differentiation toward photoreceptor precursors. Nanoscale 12, 18918–18930 (2020).

      Ueta, Miho et al. “Effects of TGF‑β1 on the migration and morphology of RAW264.7 cells in vitro.” Molecular medicine reports vol. 20,5 (2019): 4331-4339.

      Ye, Haibo et al. “Bilirubin-induced neurotoxic and ototoxic effects in rat cochlear and vestibular organotypic cultures.” Neurotoxicology vol. 71 (2019): 75-86.

      Burks et al., 2013. ISGylation governs the oncogenic function of Ki-Ras in breast cancer. Oncogene. doi:10.1038/onc.2012.633

      Andersen et al., 2012. Escherichia coli uropathogenesis in vitro: Invasion, cellular escape, and secondary infection analyzed in a human bladder cell infection model. Infect. Immun. 80, 1858-1867.

      Question 1:  Can I use fluorescently-labeled phalloidin to stain F-actin in living cells?

      Answer 1:  Unfortunately, no, phalloidin cannot be used to stain F-actin in living cells.  Phalloidins are used to stain F-actin in fixed cells.  Fluorescent phalloidins only bind to the native quaternary structure of F-actin which provides a low background.  The correct fixation condition for phalloidin binding is 3.7% (v/v) paraformaldehyde in PBS for 10 min because it retains the quaternary protein structure which is necessary for high affinity binding of phalloidin.  Methanol fixation destroys the native conformation and hence is not suitable for F-actin staining with phalloidin.  To monitor actin dynamics in living cells, micro-injection of rhodamine-labeled actin (Cat. # APHR or AR05) is recommended.  Please see those datasheets for more information.

       

      Question 2:  Which of the fluorescently-labeled phalloidin is the most stable/brightest?

      Answer 2:  The brightest and most stable of the Acti-stains is Acti-stain 488 (green fluorescence; Cat. # PHDG1).  Please see the table below for additional information on all of our Acti-stains. 

      Conjugate

      Cat. #

      Wavelengths (Ex/EM)

      Brightness (AFU)

      Stability to photobleaching* (1/2 life in sec)

      Background (% of total AFU at 100 nM)

      Acti-stain™ 488

      PHDG1

      485/535

      832

      57

      5

      Acti-stain™ 535

      PHDR1

      535/585

      430

      27

      12

      Acti-stain™ 555

      PHDH1

      535/585

      551

      46

      16

      Acti-stain™ 670

      PHDN1

      640/680

      332

      18

      18

      * = measured in the absence of antifade.

       

      If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.