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Preparation and Characterization of Fluorophenylboronic Acid-Functionalized Affinity Monolithic Columns for the Selective Enrichment of cis-Diol-Containing Biomolecules
Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity ...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Mixed-Bed Affinity Chromatography: Principles and Methods
Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Immobilized Magnetic Beads-Based Multi-Target Affinity Selection Coupled with HPLC-MS for Screening Active Compounds from Traditional Chinese Medicine and Natural Products
Screening and identifying active compounds from traditional Chinese medicine (TCM) and other natural products plays an important role in drug discovery. Here, we describe a magnetic beads-based multi-target affinity selection-mass spectrometry approach for screening bioactive compounds from natural products. Key steps and parameters including activation of magnetic beads, enzyme/protein immobilization, characterization of functional magnetic beads, screening and identifying active compounds from a complex mixture by LC/MS, are illustrated. The proposed approach is rapid and efficient in screening and identification of bioa...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Macroporous Silica Particles Derivatized for Enhanced Lectin Affinity Enrichment of Glycoproteins
This chapter details procedures for (1) functionalizing macroporous silica particles with lectins, a class of proteins that have affinity for the glycan moieties on glycoproteins, and (2) utilizing the lectin-silica material for high-performance affinity chromatography (HPAC) to enrich glycoproteins from small volumes of biological sample materials. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Robotic High-Throughput Purification of Affinity-Tagged Recombinant Proteins
Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditio...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

The Strep-tag System for One-Step Affinity Purification of Proteins from Mammalian Cell Culture
The Strep-tag—or its improved version Strep-tagII—is an eight amino acid sequence that can be easily fused or conjugated to any protein or peptide of interest and that was engineered for high affinity toward streptavidin, which otherwise is widely known as a tight biotin-binding reagent. Especially in combination with immobilized Strep-Tactin, a mutant streptavidin specifically optimized toward the Strep-tagII, this system enables the facile one-step affinity purification of various biomolecules, including oligomeric and even membrane proteins. The Strep-tagII/Strep-Tactin interaction shows exquisite specificit...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Aptamer-Modified Magnetic Beads in Affinity Separation of Proteins
Aptamers are valuable alternative ligands for affinity separations. Here, we describe the aptamer-based affinity separation of His-tagged proteins using an aptamer directed against the His-tag. The immobilization of the aptamer to magnetic beads is described as well as the aptamer-based purification and proper methods for the characterization of the process. Moreover, indications for the transfer of the process to other aptamers are given. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Cell Affinity Separations on Microfluidic Devices
Separating cells from a heterogeneous sample on microfluidic devices is a very important unit operation in biological and medical research. Microfluidic affinity cell chromatography is a label-free separation technique, providing ease of operation, low cost, and rapid analysis. In this chapter, protocols for cell affinity separation in polydimethylsiloxane (PDMS)–glass microdevices and glass capillaries are described. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Specific Recognition of Supercoiled Plasmid DNA by Affinity Chromatography Using a Synthetic Aromatic Ligand
Liquid chromatography is the method of choice for the purification of plasmid DNA (pDNA), since it is simple, robust, versatile, and highly reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes toward designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential rete...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Antibody Purification from Human Plasma by Metal-Chelated Affinity Membranes
Immobilized metal ion affinity chromatography (IMAC) has been used for purification of proteins. IMAC introduces a new approach for selectively interacting biomolecules on the basis of their affinities for metal ions. The separation is based on different binding abilities of the proteins to the chelated metal ions on support. Here, N-methacryloyl-(l)-histidine methyl ester (MAH) is used as the metal-chelating ligand. Poly(hydroxyethyl methacrylate) Poly(HEMA) based membranes were prepared by photo-polymerization technique. Then, Zn2+, Ni2+, Co2+, and Cu2+ ions were chelated directly on the poly(HEMA-MAH) membranes for puri...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

One-Step Purification of Phosphinothricin Acetyltransferase Using Reactive Dye-Affinity Chromatography
Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Protein Purification by Aminosquarylium Cyanine Dye-Affinity Chromatography
Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Affinity Chromatography: A Historical Perspective
Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generati...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news

Sample Preparation for N-Glycosylation Analysis of Therapeutic Monoclonal Antibodies by Electrophoresis
There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity. The structural diversity of these attached glycans can be manifested in altered monosaccharide composition and linkages/positio...
Source: Springer protocols feed by Biochemistry - February 13, 2015 Category: Biochemistry Source Type: news

On-Chip Electromembrane Extraction for Monitoring Drug Metabolism in Real Time by Electrospray Ionization Mass Spectrometry
Sample preparation is an essential step in any bioanalytical procedure and very often the most challenging step in method development. Most of the currently used methods require a relatively large amount of sample and are time consuming. Here, we describe a new approach based on electromembrane extraction (EME) integrated in microfluidic polymer chips. This procedure is fast, requires only small amounts of sample, and may thus be used for monitoring drug metabolism and the formation of metabolites in real time. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - February 13, 2015 Category: Biochemistry Source Type: news