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Product Details
| Cat # +Size | K457-100 |
|---|---|
| Size | 100 assays |
| Kit Summary | • Fluorescence (Ex/Em: 365/410 nm) • Study and quantitate the effect of different compounds, proteins and tissue extracts on Actin polymerization and/or depolymerization • Evaluation of critical concentrations of actin polymerization in different conditions |
| Detection Method | Fluorescence (Ex/Em: 365/410 nm) |
| Species Reactivity | Not Applicable |
| Applications | • Study and quantitate the effect of different compounds, proteins and tissue extracts on Actin polymerization and/or depolymerization • Evaluation of critical concentrations of actin polymerization in different conditions |
| Kit Components | • Buffer G • Buffer P (10X) • Labeled Rabbit Muscle Actin • ATP (100 mM) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Actin, a highly conserved and abundant protein in eukaryotic cells, is one of the major components of cytoskeleton. It can be found as monomeric globular protein, called G actin or it can polymerize into filamentous actin, named F actin. Actin plays major roles in cell division, cell motility, cell signaling, organelle movement, etc. Mammals have 6 isoforms of Actin, which can be divided into 3 classes, α, β and γ. Muscle Actin is α class and all other non-muscle actins belong to β and γ -classes. Understanding the effect of different drugs, proteins, etc. on Actin Polymerization and Depolymerization is very important for understanding cellular machinery, more importantly because Cytoskeleton is a very important target for cancer therapy. BioVision’s Actin Polymerization/Depolymerization Assay Kit can be used to study the effect of different compounds, proteins and tissue extracts on Actin polymerization and depolymerization. The kit utilizes a proprietary Pyrene-labeled Actin molecule that develops a higher fluorescent signal if it undergoes polymerization. The signal can be easily detected using a fluorescence microplate reader. The assay is simple, high- throughput compatible, and can be completed in less than three hours
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Can you please provide me the protocol for tissue or cell lysate preparation?
Below is the detailed protocol:
1. Prepare an extraction buffer consisting of 20 mM Tris-HCl, pH 7.5, and 20 mM NaCl, plus any co-factors for your protein and a protease inhibitor cocktail.
2. Isolate extracts with ice-cold buffer and lyse the cells either using a 25 g bent over syringe needle or other device.
3. Make a 10 mg/ml protein extract with the ice-cold buffer.
4. Add 1/3rd volume of the extract to 2/3rd volume of pyrene-actin (labelled actin) to insure there is high enough concentration of protein to exert an effect on actin polymerization.
5. Prepare extract from experimental and control cells. The control cell line is very critical because the actin polymerization reaction is very sensitive to slight differences in protein concentration and salt.
1. Prepare an extraction buffer consisting of 20 mM Tris-HCl, pH 7.5, and 20 mM NaCl, plus any co-factors for your protein and a protease inhibitor cocktail.
2. Isolate extracts with ice-cold buffer and lyse the cells either using a 25 g bent over syringe needle or other device.
3. Make a 10 mg/ml protein extract with the ice-cold buffer.
4. Add 1/3rd volume of the extract to 2/3rd volume of pyrene-actin (labelled actin) to insure there is high enough concentration of protein to exert an effect on actin polymerization.
5. Prepare extract from experimental and control cells. The control cell line is very critical because the actin polymerization reaction is very sensitive to slight differences in protein concentration and salt.
