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40 Current white paper about the topic fluorescence

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Characterizing Biotherapeutics with HPLC

Smart Solutions for large Molecules

24-11-2010

As demonstrated here, there are a broad range of HPLC applications used in development, production, validation and release of protein therapeutics for human use. Some of the applications mentioned above are mandatory when submitting a new biopharmaceutical to approval at the regulatory agencies. The introduction of the first so-called biosimilars in Europe further increased the demand for highly efficient analysis methods. The continuous development of new stationary phases for the common modes of biochromatography, such as size exclusion, ion exchange and hydrophilic interaction chromatography support biochemists in increasing lab productivity and establishing high-throughput HPLC analysis not only for small molecules but also for large biopolymers...

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Membrane Fluidity Measurements Using UV Fluorescence Polarization and the POLARstar Omega

23-06-2010

The POLARstar Omega microplate reader from BMG LABTECH was used to study membrane fluidity via fluorescence polarization in 96-well format. The method allows for the monitoring of large scale alterations in membrane fluidity (such as phase transitions), as well as more subtle changes in lipid dynamic. The change in polarization caused by dibucaine and propranolol were investigated in both liposomes and mitochondrial membranes...

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alamarBlue® Assay for Assessment of Cell Proliferation using the FLUOstar OPTIMA

23-06-2010

The alamarBlue® cell proliferation assay is based on a reducing environment that indicates metabolic active cells. Active cells are able to convert resazurin into the resorufin. Resazurin is blue and non-fluorescent whereas resorufin is red and highly fluorescent; therefore a fluorescent and a colorimetric detection are possible. This application notes focuses on the quantification of cell proliferation in a Human Osteoblast cell culture by measuring alamarBlue® fluorescence on the FLUOstar OPTIMA microplate reader...

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Application of the Amplifluor® SNPs Genotyping System using a FLUOstar OPTIMA microplate reader

16-06-2010

The detection of SNPs (single nucleotide polymorphisms) is an important tool for researchers in order to find mutations in DNA sequences. In this application note we describe a homogeneous fluorescence assay based on PCR. The Amplifluor® SNPs HT Genotyping System from Millipore was used to screen a number of samples with the help of the FLUOstar OPTIMA multidetection microplate reader from BMG LABTECH...

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High-Throughput Cardiac Gene Expression Analysis Using the Universal ProbeLibrary and the Novel LightCycler® 1536 Real-Time PCR System

25-01-2010

Although congenital heart malformations are the most common birth defects in humans, the underlying patho­mechanisms remain widely unknown. Through linkage analyzes and candidate-gene approaches, several gene mutations causing congenital heart defects (e.g., CITED2, GATA4, NKX2-5) have been identified. However, most heart malformations display variable expressivity and pene­trance, which indicates a multifactorial and multigenic basis. This suggests that the regulatory context of transcription factors plays an important role in the manifestation of the defects. In order to better understand the molecular pathways in cardiogenesis and disease we studied the global genetic network that is deregulated in malformed hearts...

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LightCycler® 480 and 1536 Real-Time PCR Systems - Powerful Solutions for Different Throughput Levels

A Cross-Platform Comparison

09-12-2009

During the past few years real-time PCR has been proven to be a key technology for gene expression analysis, for instance for gene expression profiling and array verification studies and for the genotyping of single nucleotide polymorphisms, both in industrial and public research, with major impact on the fields of drug discovery, molecular medicine and personalized health care...

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Enzyme Kinetic Measurements for a Combinatorial Library of Inhibitors of Pseudomonas Elastase

25-11-2009

Pseudomonas elastase (LasB) is a key drug target in various chronic and intractable infections. A library of 160 potential, in-house synthesised, elastase inhibitors were screened using a fluorescence assay with the internally quenched substrate Abz-Ala-Gly-Leu-Ala-Nba. Enzymatic kinetic measurements were done on a FLUOstar OPTIMA and Km and Vmax values were calculated for each inhibitor using the MARS Data Analysis software...

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ORAC Assay Performed on the POLARstar Omega and PHERAstar FS microplate reader

15-09-2009

In all oxygen consuming cells, metabolism and oxidative stress generate several intermediates and byproducts that are collectively known as reactive oxygen species (ROS). ROS are necessary intermediates in the human body, but they are also involved in the aging process and in the development of many degenerative diseases, including cancer, heart disease, Alzheimer's and Parkinson's. Herein we describe the determination of the antioxidant capacity of different fruit juices using the ORAC assay; a standardized method that uses Trolox®, a water-soluble analogue of vitamin E, as a standard substance...

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Adaptation of SmartAmp-2 Assays for Microsatellite Copy Number Polymorphism Detection on the LightCycler® 480 Instrument

07-09-2009

The Smart Amplification process, version 2 (SMAP-2), also known as SmartAmp-2, is a rapid polymorphism detection system based on a unique asymmetric primer design, including a folding primer (FP) and a turn-back primer (TP) [1]. Combined with Thermus aquaticus MutS (Taq MutS) protein in an isothermal amplification procedure, the technique enables suppression of mis-amplification of highly related sequences (e.g., a wild-type sequence versus an SNP variant), minimizing background amplification. The Taq MutS protein binds to mis-matched nucleotides in erroneously primed dsDNA and prevents further amplification by blocking the dissociation of DNA duplexes having a mis-match by DNA polymerases having strand displacement activity. SmartAmp-2 assays have been used for detection of various SNPs and mutations, e.g., in ALDH2 and EGFR genes [1,2]. In the study presented here, we investigated whether this assay principle could be adapted for detection of microsatellite polymorphisms showing copy number differences in...

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High-Throughput Analysis Using the Novel LightCycler® 1536 Real-Time PCR System

07-09-2009

An important trend in biological and biochemical analysis over the past 15 years has been miniaturization and parallelization of analytical procedures. Large-scale high-throughput analysis of gene expression or genetic polymorphisms is an essential element of modern functional genomics, where individual samples are screened against many thousands of target genes. According to its superior sensitivity and accuracy, qPCR is a well-established gold standard for precise profiling of gene expression levels or genotyping of known SNPs...

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