Acti-stain 488 phalloidin

$0.00

SKU

PHDG1

Datasheet

MSDS

Amount

300 Slides (Cat. # PHDG1-A) + $239.00

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Details

Product Uses Include

Fluorescent staining of actin filaments in fixed tissue sections and tissue culture cells preparations. Note: Unlike many actin antibodies, Acti-stain™ 488 phalloidin binds only to F-actin resulting in low background fluorescence. Furthermore, binding of F-actin by Acti-stain 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 and 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 green 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™ 488 phalloidin-labeled actin filaments retain many functional characteristics of unlabeled actin including their ability to interact with myosin. Actin-stain™ 488 phalloidin is supplied as an orange solid.

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

Click Here for Detailed Methods

Example Results and Specifications

Figure 1. Actin Stress Fibers stained with Acti-stain™ 488 in a Swiss 3T3 cell.

Swiss 3T3 cell stained with anti-vinculin (red), Dapi (blue nucleus) and F-actin is stained with Acti-stain™ 488 (green F-actin, Cat.# PHDG1).

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

Absorbance and fluorescence scan of Acti-stain™ 488. Labeled phalloidin was diluted into methanol and its absorbance and excitation spectra were scanned between 350-750 and 500-750 nm, respectively. Absorbance peaks at 500 nm and fluorescence at 550 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

Conjugate	Cat.#	Wavelengths (Ex/Em)	*Brightness (AFU)**	Stability to photobleaching(half life in seconds)**	Background (% of total AFU at 100nM)**	Affinity (Kd in nanomolar)
Fluorescein-phalloidin	na	485/535 FITC filter set	432	6	22	72 +/-12
Acti-stain™ 488	PHDG1	485/535 FITC filter set	832	57	5	55 +/-8
Acti-stain™ 535	PHDR1	535/585 TRITC filter set	430	27	12	36 +/-7
Acti-stain™ 555	PHDH1	535/585 TRITC filter set	551	46	16	63 +/-8
Acti-stain™ 670	PHDN1	640/680 Cy5 filter set	332	8	18	50 +/-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.

About

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

Citations

Author	Title	Journal	Year	Article Link
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Fu, Jing et al.	The checkpoint inhibitor PD-1H/VISTA controls osteoclast-mediated multiple myeloma bone disease	Nature Communications	2023	ISSN 2041--1723
Sun, Hua et al.	Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy	Kidney360	2023	ISSN 2641-7650
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Murtaza, Nadeem et al.	Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease-relevant pathologies	Cell Reports	2022	ISSN 2211--1247
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Faqs

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	8	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.

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Acti-stain 488 phalloidin

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