Cat. # BK001

Product Uses Include

• To determine whether a protein binds to filaments or monomers of actin.
• To determine whether a protein bundles F-actin
• To test various conditions (e.g. pH optima) or requirements (e.g. divalent cation requirement) for binding to actin.
  
  

Introduction
The actin binding protein spin-down assay kit provides G- or F-actin plus positive (α-actinin ) and negative (Bovine Serum Albumin, BSA) binding control proteins. Actin binding occurs when there is an affinity for any site of actin. F-actin binding can be measured by using a spin down method. In this way centrifugation is used to separate F-actin from G-actin by differential sedimentation. F-actin binding proteins will co-sediment with actin filaments and form a pellet at the bottom of the centrifugation tube. Proteins with F-actin bundling activity can be detected since they will cause the F-actin to sediment at lower centrifugal forces than normal (14,000 x g vs 150,000 x g). F-actin severing proteins, G-actin binding proteins or non-actin binding proteins will stay in the supernatant. Severing proteins will be expected if more G-actin remains in the supernatant than in the negative control sample, and this activity should be tested further by measuring F-actin length distributions before and after adding the test protein. G-actin binding proteins can be measured by adding the test protein to G-actin and inducing polymerization, if the test protein sequesters G-actin then during the spin-down assay more actin will be left in the supernatant compared with the control.

Actin can exist in two forms: Globular subunit (G-actin) and Filamentous polymer (F-actin) (See the About Actin page for more information). Both forms of actin interact with a plethora of proteins in the cell. To date there are over 50 distinct classes of Actin-Binding Proteins (ABPs), and the inventory is still far from complete. Actin Binding Proteins allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals and are integral to cytoskeletal involvement in many cellular processes. These include cell shape and motility, muscle contraction, intracellular trafficking, cell pathogenesis and signal transduction.

This kit contains skeletal muscle actin (Cat. # AKL99). The same kit is also available with non-muscle actin (Cat. # APHL99), see Cat. # BK013. The non-muscle actin spin-down kit may be more appropriate to use to study actin binding proteins from non-muscle tissues.

Kit contents
The kit contains sufficient materials for 30-100 assays depending on assay volume. The following reagents are included:

1. 8 x 250 µg Skeletal muscle actin (Cat. # AKL99).
2. α-Actinin, positive control (Cat. # AT01).
3. BSA, negative control
4. General Actin Buffer (Cat. # BSA01).
5. Actin Polymerization Buffer (Cat.# BSA02)
6. F-actin Cushion Buffer.
7. ATP, 100 mM (Cat. no. BSA04)
8. EGTA, 0.5 M
9. MgCl₂, 100 mM
10. Tris-HCl pH 6.5, 1.0 M
11. Tris-HCl pH 7.5, 100 mM
12. Manual with detailed protocols and extensive troubleshooting guide.
    
    

Equipment required

1. Centrifugation set-up capable of 150 000 xg at 4°C and 24°C, 50 -200 µl volume capacity.
2. SDS-PAGE system.
3. Detection system for protein of interest (coomassie is good for purified proteins, Western blot or silver stain for less pure or low abundance test proteins).
4. Gel scanner for densitometric determinations.
   
   

Example results
The actin binding protein spin-down assay kit was used to study the actin binding and bundling activities of α-actinin (cat. # AT01). α-Actinin was shown to bundle F-actin and make the F-actin sediment at 14,000 x g (Fig. 1).

Figure 1. Actin bundling assay using kit BK001. F-actin was incubated alone or together with α-actinin or BSA. Bundled F-actin was pelleted by a 14,000 x g centrifugation and pellets (P) and supernatants (S) were run on an SDS-PAGE gel. Only in the presence of the F-actin bundling protein α-actinin is actin pelleted at this centrifugation speed.