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Analysis Biomolecular Interactions of Ku70 Domain β-barrel with HBoV1 NP1 by BLI (CAT#: STEM-MB-0197-CJ)

Introduction

Human bocavirus 1 (HBoV1) belongs to Primate bocaparvovirus 1 in the genus Bocaparvovirus of the Parvoviridae family. HBoV1 was first identified in 2005 by a large-scale virus screening of nasopharyngeal specimens of children with respiratory illness, and was later confirmed to be an emerging human pathogen that causes lower respiratory tract infections in young children worldwide. HBoV1 expresses one large nonstructural protein (NS1), four small nonstructural proteins (NS2, NS3, NS4, and NP1), one small noncoding RNA (bocavirus-encoded small RNA, BocaSR), and three viral capsid proteins (VP1, VP2, and VP3) from a single precursor mRNA (pre-mRNA) via alternative splicing. NS1, NP1, and BocaSR are essential for DNA replication of HBoV1. Among the nonstructural proteins, the expression of the 25-kDa NP1 is a unique feature of bocaparvoviruses. Minute virus of canines (MVC) NP1 was the first bocaparvoviral NP1 identified to play an important role in viral DNA replication and viral pre-mRNA processing through interacting with cleavage and polyadenylation specificity factor 6 (CPSF6). HBoV1 NP1 also interacts with CPSF6 and regulates polyadenylation of capsid protein-encoding mRNA. Moreover, the transport of HBoV1 NP1 to the nucleus is escorted by CPSF6. Importantly, HBoV1 NP1 colocalizes with replicating viral DNA in the viral DNA replication centers.




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: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: HEK293 Cells, HEK293T Cells, Dulbecco's modified Eagle's medium (DMEM)