Application Notes

Application Note 101: Fast Kinetics of Conformational Changes in Proteins

Understanding conformational changes in proteins and other biomolecules is important because of their critical roles in many biological functions and processes. These conformational changes are often small and fast, making it difficult to measure and monitor. Surface Plasmon Resonance (SPR) is capable to detect rather small conformational changes in surface bound molecules [1]. However, most SPR setups are not suitable

Application Note 102: Electrochemical SPR: Redox-Induced Protein Conformational Changes

Both electrochemical and surface plasmon resonance (SPR) techniques measure various processes taking place at or near an electrode surface. Combining the two techniques allows one to obtain new insight into these interfacial processes. One example is redox-induced conformational changes in surface-bound protein molecules. However, most SPR setups are not suitable for monitoring fast kinetics or small thickness variations

Application Note 103: Electrochemical SPR: Metal Deposition/Stripping and Reorganization of Electroactive Organic Thin Films

In addition to studies of redox-induced conformational changes of surface-bound proteins (cf. Application Note #102), electrochemical SPR can be used to quantify the amount of metal electrodeposited onto a surface and reorganization of organic thin films upon redox reactions. A BI-SPR 1000 was used to quantify the amount of Cu deposited and stripped during Cu2+ reduction/Cu oxidation in a CuSO4 solution

Application Note 104: Flow-Injection SPR: Sensitive Determinations of Heavy Metal Ions

Flow-injection SPR has been demonstrated as a viable alternative for sensitive detection of heavy metal ions at trace levels [1-4]. The impetus behind using SPR for elemental analysis stems from its high sensitivity, simplicity, compact design (for possible field-based work), and universal detection mechanism (e.g., any elemental species adsorbed onto the SPR sensor can cause a detectable signal). The challenge, however, is to prepare a chemically or

Application Note: 105 – Protein/Drug Interaction: Ferulic Acid and Bovine Serum Albumin

SPR has been demonstrated to be a powerful optical technique for bioaffinity studies at the solid/solution interface. In general, SPR measures the change in refractive index originated from the binding of a solution species with a molecule pre-immobilized onto the SPR sensor chip. The advantages of SPR include its simplicity, high sensitivity, obviation of sample labeling, and amenability for real-time analysis

Application Note 106: Flow Injection-SPR: Sensitive and Sequence-Specific DNA Assays

SPR (SPR) has been demonstrated as a powerful technique for rapid, sensitive, and label-free genetic analysis [1-5]. When the sensor surface is coated with a single sensing (probe) DNA, SPR can be used for both affinity binding studies (i.e., kinetic measurements) and concentration detection of a target DNA. However, the concentration detection levels for SPR and SPR imaging are typically at low nanomolar (nM) [3, 4],

Application Note: 107 – Binding Kinetics Analysis with SPR: Interaction between Bovine Serum Albumin (BSA) and Anti-BSA

In restriction digestion, BSA has been used to stabilize enzymes during DNA digestion. It is also widely used as a biomolecule to block active sites on surfaces. Formation of the anti-BSA/BSA immune complex is relevant to studies of the receptor site of the red blood cells..

Application Note: 108 – SPR as a Chromatographic Detector: Separation and Label-Free Detection of Biomolecules

In recent years, SPR has been used as an alternative detector for monitoring elution of a variety of species (e.g., polysaccharides and proteins [1-4]) out of liquid chromatographic (LC) columns. In addition to simplicity and fast speed the SPR detector can detect any eluent, as long as its index of refraction is different from that of the carrier buffer. The open design of the BI-SPR instruments allows users to conveniently connect

Application Note: 109 – Application of SPR to Bacteriology: Endotoxin/Protein Interaction Studies

Endotoxin (commonly referred to as lipopolysaccharide in bacteriology) is associated with the outer membrane of Gram-negative bacterial pathogens such as Escherichia coli, Salmonella, Shigella, and Pseudomonas [1-2]. The interaction between endotoxin and bacterial cell surface is schematically depicted in Figure 1. Endotoxin elicits a series of pleiotropic effects on cells or organisms and is therefore harmful to most mammals

Application Note: 110 – Studying Protein Adsorption Properties with SPR

Surface Plasmon Resonance (SPR) can be applied as a convenient, sensitive and label-free technique to study various surface phenomena. One such example is the Vroman effect exhibited by protein adsorption onto surfaces. This important effect arises from the fact that protein adsorption capability onto a surface depends on motility, which is intimately related to its molecular weight. In general, a high molecular weight (HMW) protein adsorbs

Application Note: 111 – Measuring Surface Charge Density: A New Application with SPR

Surface charge density is a basic quantity that is directly relevant to many phenomena, from surface interactions to DNA hybridization. Measurement and quantification of surface charge density can lead to a better understanding of biomolecular interactions on surfaces. To date, different techniques have been developed to measure surface charge density, including potentiometric titration, atomic force microscopy and reflection interference

Application Note: 112 – Detection of Wild-Type and Mutant p53 Proteins in Cancer Cell Lysates

This application note describes the simultaneous SPR detection of wild-type and mutant p53 proteins in cancer cell laysates. p53 is a transcription factor (i.e., DNA-binding protein) that plays an important role in DNA repair and tumor suppression by inhibiting the growth of tumor cells through eliciting either cell-cycle arrest or apoptosis [1-3]. In solution, p53 molecules tetramerize at their C-termini and the resultant p53 tetramer can bind

Application Note: 113 – Flow-Through Electrochemical Surface Plasmon Resonance (SPR): Detection of Intermediate Reaction Products

SPR is sensitive to various processes taking place on or near a sensor chip. The SPR sensor chip can also simultaneously serve as a working electrode for electrochemical measurements. Combining electrochemical with SPR measurements has led to the development of Electrochemical SPR (EC-SPR). To date, EC-SPR has been used in the analysis of trace metal ions, detection of surface bound redox species, electrochemical polymerization

Application Note 114: SPR Binding Affinity Determination of Novel Peptide Inhibitors to the Innate Immune activator C1q

The complement system is an essential component of the human innate immune system, playing a critical role as a defense mechanism against invading pathogens, priming adaptive immune responses and helping to remove immune complexes and apoptotic cells. Three different pathways comprise the complement system: the classical, lectin and alternative pathways [1]. While the complement system plays a central role

Application Note 115: SPR Assay of Clinical Alzheimer Disease Samples: Amyloid β Peptides in Cerebrospinal Fluids

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, affecting over 6.5 million people over the age of 65 in the U. S. In the senile plaques of AD patient’s brain, the major components are peptides composed of 39–43 amino acid residues (referred to as the amyloid β or Aβ peptides).[1-2] One of the hypotheses for AD neuropathology is that the misfolding and subsequent aggregation of these peptides

Application Note 116: Temperature Dependence of Enzymatic Cleaving Activity

Some biomolecules such as enzymes can exhibit much greater biological activities at physiological temperature (37.5 °C) or higher (e.g., DNA polymerase). The focus of this study is to screen for potential inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 is an enzyme that cleaves the transmembrane amyloid precursor protein to produce the amyloid beta

Application Note 117: Real-time Monitoring Biomarker Expression of Carcinoma Cells

Vascular endothelial growth factor (VEGF) is an angiogenic signal protein biomarker produced by oxygen-hungry cells to stimulate the growth of blood vessels.[1] It binds to specialized receptors on the surfaces of endothelial cells and directs them to grow new blood vessels during embryonic development. Certain types of tumor cells produce abnormally large amounts of VEGF or block the action of angiogenesis inhibitors

Application Note 118: SPR for Food Safety and Monitoring: Melamine in Milk Samples

In 2008, the illegal adulteration of infant milk products with melamine (structure shown in FIG 1) led to an outbreak of adverse kidney and urinary effects in thousands of children in China.[1] Clandestine addition of melamine in milk products was to boost the detectable protein contents. Because of the severe impact of melamine on human health, the World Health Organization (WHO)

Application Note 119: Small Molecule Detection by Surface Plasmon Resonance (SPR)

A major advantage of SPR technology is that the detection does not require the analyte to be labeled, such as with a fluorescent molecule or a redox-active tag. This is because SPR directly detects changes in refractive index resulting from changes in mass at the sensor chip surface. For researchers interested in pharmacology and pharmacokinetics or in general pharmaceutical research or drug discovery, this capability of label-free detection is particularly attractive.

Application Note 120: Electrochemical SPR Monitoring of Drug Interactions with Live Cancer Cells

By combining SPR with electrochemical methods, many new capabilities and applications are now possible. This application note describes the measurement of apoptosis of cancer cells (HepG2) after treatment of Daunorubicin (DNR), an anti-cancer drug commonly used to treat specific types of leukemia.

Application Note 121: Protein Interactions in the Mycofactocin Biosynthetic Pathway

Protein-protein interactions are essential for many biochemical transformations and cellular processes. However, traditional methods of detecting these Surface plasmon resonance (SPR) is capable of analyzing protein-protein interactions kinetically while consuming little protein sample and does not rely on absorbance/fluorescence properties of proteins. This application note describes the SPR analysis of label-free protein-protein interactions found in the mycofactocin biosynthetic pathway.

Application Note 122: Surface Plasmon Resonance Microscopy for Multiple and Single Cell Membrane Binding Kinetics Studies

Surface Plasmon Resonance (SPR) is a powerful technique for measuring the binding kinetics of biomolecular interactions in a real-time and label-free manner [1]. In traditional SPR assays, the target molecule is extracted and purified from the cell and immobilized onto the sensor surface for measurement of the interaction kinetics between the target and the drug candidate.

Application Note 123: Surface Plasmon Resonance Measurement of Protein-Peptide Interaction Using Streptavidin Sensor Chip

Streptavidin (SA) sensor chips are commonly used for kinetic interactions of biotinylated ligands. Streptavidin is a tetrameric molecule with an extremely high affinity towards biotin and, because of its binding capacity, reproducibility and chemical resistance,streptavidin is widely used for bioconjugation. SA chips facilitates detection of small molecules, as well as the use of harsh regeneration […]

Application Note 124: Quantifying Molecular binding to Membrane Proteins on Individual Cells with Surface Plasmon Resonance Microscopy

SPRm 200 System is the world’s first commercial Surface Plasmon Resonance Microscopy (SPRM). It combines the high spatial resolution of optical microscopy with the powerful sensing capability of SPR, making it possible to measure the binding of molecules label free with membrane proteins on single intact cells for the first time. Cells are incubated on […]

Application Note 125: Lectin-Glycoprotein Interactions with SPR Microscopy

Membrane proteins are involved in many biological processes, such as signaling between cell’s internal and external environments, transport of ions and molecules and catalysis of chemical reactions [1] and they are also the targets of more than 60% of all drugs. [2]  Determining the binding of ligands or drug molecules to membrane proteins is critical […]

Application Note 126: GPCR Binding Assays with SPR Microscopy

G-protein coupled receptors (GPCR) are integral membrane proteins that transmit signals from external stimuli to the cell interior. They play key roles in many cellular processes, such as sensorial, hormonal, metabolic, immunological, and neurotransmission processes(1), and are the most popular membrane protein drug targets for a wide range of diseases, including cancer, immune and inflammatory disorders […]

Application Note 127: Quantifying Antibody Binding to Membrane Proteins on Single Cells with SPR Microscopy

Monoclonal antibody (mAb) therapies have become established methods for treating cancer, autoimmune disorders, asthma and many other diseases. MAb drugs represent approximately half of the total sales of all biopharmaceutical products (1) and over 60% of them target membrane proteins on the cell surface. (2)  However, quantifying mAb-membrane protein binding affinity on single cells has […]

Application Note 128: SPR Microscopy for Acid-Sensing Ion Channels

Acid-sensing ion channels (ASICs) are voltage independent cation channels, which are expressed in both central and peripheral neurons.[1]  Four genes that encode six ASIC subunits have been identified in mammals (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4) and these ASIC subunits assemble into homo and hetero-trimmers with singular biophysical properties.[2]  ASICs are important drug targets […]

Application Note 129: AZ1395 small molecule targeting GPR39

Membrane proteins play critical roles in cellular communications and are the most popular drug targets, accounting for over a half of the FDA approved drugs. One particularly important example of membrane proteins is G protein coupled receptors (GPCRs), which constitute the largest and most diverse protein family in the human genome with over 800 members […]

Application Note 130: Measuring molecular binding kinetics with SPRM Impedance

SPR Microscopy (SPRM) is a powerful tool for measuring the binding kinetics of ligands or drug compounds with membrane proteins, including GPCRs. It has many other unique features that lead to new applications.  A recent example is the integration of SPRM with electrochemical methods for molecular binding kinetics quantification. Researchers at Arizona State University and […]

Application Note 131: SPR Microscopy for Live Suspension Cells

SPRm 200 system is the world’s first commercial Surface Plasmon Resonance Microscopy (SPRM) which combines the high spatial resolution of optical microscopy with the label-free sensing capability of SPR.   With this instrument, drug molecules binding with membrane proteins on single whole cells can be directly detected and quantified. In this application note, we describe the […]

Application Note 132: SR142948 Antagonist Binding to NTS-1 Receptor Using SPR Microscopy

Neurotensin receptor belongs to the family of G protein-coupled receptors (GPCR), which has 424 amino acids with 7 putative transmembrane domains.(1)  Neurotesin receptor 1 (NTS-1) mediates multiple functions of neurotensin, such as hypotension, hyperglycemia, hypothermia, antinociception, and regulation of intestinal motility and secretion.(2) Despite the importance, a suitable assay that can directly measure GPCR-drug interactions […]

Application Note 133: Interaction of Protein Phosphatase 1 with its Muscle Glycogen– targeting Regulatory Subunit Measured by SPR

Glycogen is the primary storage form of glucose. Glycogen synthesis and breakdown are tightly controlled by glycogen synthase (GYS) and phosphorylase, respectively. The enzyme responsible for the process is protein phosphatase 1 (PP1). The dephosphorylation of glycogen synthase and phosphorylase a (glycogen phosphorylase phosphorylated on Ser14 is commonly referred to as phosphorylase a) by PP1 […]