Binding Proteins

Actin interacts with a multitude of proteins in the cell
It is widely known that F- and G-actin interact with a multitude of proteins in the cell (14). At present there are over 150 known actin binding proteins (ABPs), which accounts for approximately 25% of cellular protein. ABPs impart a great deal of functional diversity upon the actin cytoskeleton which is involved in processes such as muscle contraction, lamellopodia extrusion, cell locomotion, cytokinesis and cytoplasmic streaming. Investigating how these processes regulate actin filament function and dynamics is an important and ever growing field of research.

Actin binding proteins maybe isolated and studied by using biochemical, genetic and immunological approaches. Because there are over 50 types of actin binding proteins the methods used to identify them and their function/activities are also numerous and varied. Actin affinity columns are often used to affinity purify ABPs whether they are F- or G-actin binding. A simple assay to show that a potential ABP affects actin dynamics can be carried out by exploiting polymerization enhancement of pyrene actin fluorescence (see measuring actin polymer section). Studies of this nature have been instrumental in identifying novel actin binding proteins. There are several reviews worth reading on the possible approaches to identify and quantify novel actin binding proteins (15,16,17)

Cytoskeleton's actin products have been cited hundreds of times over the past 18 years. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.



Question 1: Which Actin Binding Protein (ABP) is useful as an F-actin binding control? 

Answer 1: The actin binding protein alpha-actinin (Cat. # AT01) is an F-actin binding and cross-linking protein, making it useful as a control for F-actin binding studies. It's Kd is 1 µM which means that 50% binding occurs at this concentration. Therefore we aim to use alpha-actin at 2 µM as a positive control in binding assays. A 2 µM concentration of alpha-actinin is equivalent to 0.23 µg/µl in the binding reaction. Also alpha-actinin is sensitive to pH where its highest affinity occurs at pH 7.0 .

Question 2: Does Cytoskeleton's myosin or heavy meromyosin have F-actin motility activity? 

Answer 2: Unfortunately we do not sell motility grade myosin or heavy meromyosin for actin motility assays.  We found that quality control tests were very difficult to reproduce and therefore we could not adequately ensure the quality of reagents. Therefore the myosins (Cat. # MY02 and MY03) and the Heavy meromyosin protein (Cat. # MH01) are not recommended for F-actin motility assays. Customers who have tested these reagents have not had good success with motility and therefore we do not recommend purchasing samples to test either. If motility grade actin or heavy meromyosin is desired we recommend following the original protocol (Kron & Spudich, 1985) and freezing 100 µl aliquots in liquid nitrogen to ensure stability.

    Reference:  S.J. Kron  and J.A. Spudich, 1985. Fluorescent actin filaments move on myosin fixed to a glass surface. PNAS, 83, 6272-6276.

Please contact tservice@cytoskeleton.com with questions regarding these products.