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Analysis Biomolecular Interactions of G-actin with Immobilized GST–ArhGAP12 by BLI (CAT#: STEM-MB-0188-CJ)

Introduction

Spatial and temporal control of the actin cytoskeleton in response to local signalling or mechanical cues plays a critical role in development and disease. Underpinning it is the regulation of actin treadmilling, the dynamic transition between the monomeric (G-actin) and polymerized (F-actin) forms of actin, which is controlled by small GTPases of the rho family. One connection between the cytoskeletal dynamics and control of protein function is provided by the RPEL proteins, which act as G-actin sensors. Their regulatory domains contain RPEL motifs (Pfam PF02755), short polypeptide sequences that bind G-actin.




Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Cytoskeletal dynamics

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Colorimetric rhoGAP assay kit
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