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Analysis Biomolecular Interactions of P58 IPK with NP and Hsp40 by BLI (CAT#: STEM-MB-0175-CJ)

Introduction

The P58IPK, an endogenous PKR inhibitor from the tetratricopeptide repeat (TPR) family, contains nine tandemly arranged repeats (TPR1-9) of 34 amino acids at the N-terminus. The homology between its C-terminal domain and the J domain of the DnaJ heat shock proteins also makes P58IPK a member of the HSP family of proteins. The P58IPK can inhibit PKR by direct binding and interruption in PKR dimerization. The PKR binding domain of P58IPK has been mapped to its TPR6 motif. Both NP and Hsp40 share a common binding site on P58IPK. And NP competes with Hsp40 for binding to P58IPK, will lead to the dissociation of Hsp40-P58IPK complex.




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

Immunology/Inflammation; Virology

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: Gator® Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Human embryonic kidney 293T (HEK293T), Dulbecco's Modified Eagle's Medium (DMEM)