Fluorescent actin assays for cytoskeleton dynamics

Atto-550 labeling in Immuno-Fluorescence tebu-bio

Actin dynamics are constantly controlled by numerous types of physiological elements (Actin Binding Proteins, Calcium levels, pH, extracellular stimuli, intracellular signaling pathways…). Understanding and deciphering cytoskeleton actin dynamics and their molecular modulation is challenging. Nowadays, fluorescent Atto-actin conjugates are available for these purposes. Let’s take a moment to review actin research tools for actin filament analysis.

There are various in vitro methods to vizualise and measure actin assembly kinetics and actin molecular interaction.

# 1 – Actin filament vizualisation in vitro

In these experiments, actin filaments are stained with fluorescent dyes (ex. Phalloidins) in fixed cells / tissues. Such actin staining methods are designed to see actin filament organization and potentially analyse their interaction with Actin Binding proteins (ABPs).

Actin can also be detected and vizualised with classical immuno-histochemistry by using highly specific primary antibodies; detection is made with fluorescence- or HRP-conjugated secondary antibodies.

# 2 – Dye conjugated actin for actin assembly dynamics

Dye-conjugated actin (ex. Pyrene) brings additional information compared to fluorescent actin dyes and actin-specific antibodies.

When actin is directly conjugated with a dye, in vitro monitoring of actin (de)polymerisation kinetics over time is possible. For example, Pyrene fluorescence-based methods are compatible with cell and tissue extracts but also with recombinant or purified proteins interacting with the cytoskeleton actin network, making it possible to investigate the effects of biomolecules of interest on actin filamant formation. Thus, the fluorescent readout of monomer pyrene actin is enhanced during its polymerization into filaments. Stringent quality control ensures that pyrene F-actin has a 7-12 fold fluorescent enhancement over Pyrene G-actin.

Despite the robustness of such dye-actin conjugates, there is now a strong need for high definition images, ultra resolutive and sensitive methods to attain single molecule imaging. Recent advances in the field of fluorescent dyes bring new perspectives regarding cell cytoskeleton actin network structure and dynamics studies.

#3 – Fluorescence Atto-actin – the actin assay generation 2.0

Atto dyes are the next generation of bright fluorescence markers. Because of their unique structure and properties, they bring strong absorption capacities, high fluorescence quantum yield and extreme photostability to in vitro assays. Conjugated with biomarkers of interest, these unique characteristics make Atto fluorescent dyes outstanding research tools for high definition imaging systems and microscopy analysis.

tebu-bio provides actin proteins conjugated with Atto dyes. Depending on the colour selected in the Atto-dye spectrum, it is possible to fully control experimental conditions to obtain high resolution actin-based microscopy images while avoiding non specific absorption and sample autofluorescence (red spectrum).

Atto dyes spectrum and table of conversion for Atto-actin conjugated tebu-bio

Atto dyes spectrum and corresponding sources of light for tebu-bio Atto-actin.

 

tebu-bio’s experts have selected 4 Atto dyes (390, 488, 550 and 647) from the spectrum to cover the major requirements when analysing actin polymerization and cytoskeleton dynamics. These Atto-actin conjugates from tebu-bio are prepared from purified G-actin conjugated with activated Atto dye to Lysine residues. They are validated to exhibit native actin polymerization properties.

  1. Atto390-Actin – G-actin from skeletal muscle rabbit – 1x100µg  (cat. nr 465001-100UG)Atto-550 labeling in Immuno-Fluorescence tebu-bio
  2. Atto488-Actin – G-actin from skeletal muscle rabbit – 1x100µg (cat. nr 465002-100UG)
  3. Atto550-Actin – G-actin from skeletal muscle rabbit – 1x100µg (cat. nr 465003-100UG)
  4. Atto647-Actin – G-actin from skeletal muscle rabbit – 1x100µg (cat. nr 465004-100UG)

Atto-actins meet your expectations when high definition and single molecule imaging are required. They are thus compatible with all microscopy methods including  TIRF, STED…

Note: Actin polymerization inhibitors such as Cytochalasin D (10-2071) or more potent small molecules (Latrunculin A (10-2254) and Latrunculin B (10-4303)) are ideal tools for studying actin cellular organization.

Want to use these highly fluorescent and photostable Atto-actins for your cytoskeleton studies?

Atto-conjugated actins are the 2.0 generation tools for molecular imaging of cytoskeleton actin filaments . You might like to visit tebu-bio’s web site to learn more about these actins or contact their experts in cytoskeleton molecular modeling to further discuss your needs.

 

Philippe Fixe, PhD
Written by Philippe Fixe, PhD
Philippe Fixe is Marketing Director at tebu-bio, passionate about innovation and R&D in Life sciences, Biotechnology, Medical research, Drug discovery, and also a keen photographer.