4. To quantify protein and other biological molecules
To detect the interaction of drug and protein
To detect the interaction of hormone and protein
To detect interactions of other macromolecules and protein
To interface mass spectrometer provides discovery based proteomics study.
Objectives
5. Surface plasmon resonance
Surface Plasmon Resonance (SPR) spectroscopy is an optical method which
is rapidly developing technique for the study of ligand binding interactions
with membrane proteins, which are the major molecular targets for drug
binding in drug discovery.
• It measures change in refractive index of the medium in close vicinity o
a metal surface which is use to monitor binding of analyte molecules to
receptor.
• It is optical biosensor technique based on phenomenon of Evanescent
waves.
• Evanescent waves are the waves travelling in the medium which
undergo total internal reflection at the boundary.
6. Methadology
Wash the surface of
the chip with the
buffer
Allow analyte to
pass through same
running buffer
Analyte start
interacting with
immobilized
receptors
When all receptors
are occupied
,steady state occurs
Allow buffer to pass
again which causes
dissociation of
analyte
Use the same chip
by thoroughly
washing with the
running buffer
7. Baseline remains straight
When analyte interacts
association phase is observed
Kon or Ka = Association rate
When all receptors are occupied
steady state is observed
Stochastic steady state
When analyte dissociates
dissociation phase is observed
Koff or Kd = Dissociation rate
8. Applications of SPR
In drug discovery and development
Cell signaling
Diagnostic assays
To identify food contaminants
To study energrtics
10. Cancer
• SPR biosensors are used to screen active ingredients that bind to the tumor
necrosis factor receptor R1. This ubiquitous receptor can activate the transcription
factor NF-κB, mediate apoptosis, and function as a regulator of inflammation.
• In this study SPR-bound factors were recovered and analyzed further by mass
spectrometry. The compound physcion-8-O-β-D monoglucoside was identified as
binding to the tumor necrosis factor receptor.
• SPR has also been used in the detection of inhibitors of Epstein–Barr virus nuclear
antigen. Epstein–Barr virus is associated with human B cell lymphomas and certain
carcinomas. Persistence of the Epstein–Barr virus is dependent on its nuclear
antigen, EBNA1, which undergoes homodimerization.
• A Biacore SPR assay demonstrated that a small-molecule inhibitor of EBNA1
dimerization, EiK1, specifically associates with EBNA15
