Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Small GTPases in Acrosomal Exocytosis
Regulated exocytosis employs a conserved molecular machinery in all secretory cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Rab superfamilies are members of this machinery. Rab proteins are small GTPases that organize membrane microdomains on organelles by recruiting specific effectors that strongly influence the movement, fusion and fission dynamics of intracellular compartments. Rab3 and Rab27 are the prevalent exocytotic isoforms. Many events occur in mammalian spermatozoa before they can fertilize the egg, one of them is the acrosome reaction (AR), a type of regulated exocytos...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Functional Analysis of Rab27A and Its Effector Slp2-a in Renal Epithelial Cells
Polarized epithelial cells have two distinct plasma membrane domains, i.e., an apical membrane domain and a basolateral membrane domain, that are the result of polarized trafficking of proteins and lipids. Several members of the Rab-type small GTPases, which are general regulators of membrane trafficking, have been reported to be involved in the regulation of polarized trafficking in epithelial cells, but their precise role in polarized trafficking is poorly understood. In a recent study we used Madin-Darby canine kidney (MDCK) II cells as a model of polarized cells and concluded from the results that Rab27A and its effect...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Identifying a Rab Effector on the Macroautophagy Pathway
Rab GTPases are key regulators of membrane traffic. The Rab GTPase Ypt1 is essential for endoplasmic reticulum (ER)-Golgi traffic, intra-Golgi traffic, and the macroautophagy pathway. To identify effectors on the macroautophagy pathway, known autophagy-related genes (Atg genes) required for macroautophagy were tagged with GFP and screened for mislocalization in the ypt1-2 mutant. At the pre-autophagosomal structure (PAS), the localization of the serine/threonine kinase Atg1 was affected in the ypt1-2 mutant. We then used an in vitro binding assay to determine if Atg1 and Ypt1 physically interact with each other and co-immu...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Molecular Modeling of the Affinity Chromatography of Monoclonal Antibodies
Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein–ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Determination of the Kinetic Rate Constant of Cyclodextrin Supramolecular Systems by High-Performance Affinity Chromatography
The kinetics of the association and dissociation are fundamental kinetic processes for the host–guest interactions (such as the drug–target and drug–excipient interactions) and the in vivo performance of supramolecules. With advantages of rapid speed, high precision and ease of automation, the high-performance affinity chromatography (HPAC) is one of the best techniques to measure the interaction kinetics of weak to moderate affinities, such as the typical host–guest interactions of drug and cyclodextrins by using a cyclodextrin-immobilized column. The measurement involves the equilibration of the c...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Characterization of the Binding Strengths Between Boronic Acids and cis-Diol-Containing Biomolecules by Affinity Capillary Electrophoresis
The affinity of boronic acids toward cis-diol-containing biomolecules has found wide applications in many fields, such as sensing, separation, drug delivery, and functional materials. A sound understanding of the binding interactions will greatly facilitate exquisite applications of this chemistry. Traditional techniques are associated with some apparent drawbacks, so they are only applicable to a limited range of boronic acids and cis-diol-containing biomolecules. This chapter describes an affinity capillary electrophoresis (ACE) method for the characterization of the binding strengths between boronic acids and cis-diol-c...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Accurate Protein–Peptide Titration Experiments by Nuclear Magnetic Resonance Using Low-Volume Samples
NMR spectroscopy allows measurements of very accurate values of equilibrium dissociation constants using chemical shift perturbation methods, provided that the concentrations of the binding partners are known with high precision and accuracy. The accuracy and precision of these experiments are improved if performed using individual capillary tubes, a method enabling full automation of the measurement. We provide here a protocol to set up and perform these experiments as well as a robust method to measure peptide concentrations using tryptophan as an internal standard. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Analysis of Drug–Protein Interactions by High-Performance Affinity Chromatography: Interactions of Sulfonylurea Drugs with Normal and Glycated Human Serum Albumin
This report describes how HPAC can be used to provide information on the number of binding sites, equilibrium constants, and changes in binding that can occur during drug–protein interactions. This approach will be illustrated through recent data that have been obtained by HPAC for the binding of sulfonylurea drugs and other solutes to the protein human serum albumin (HSA), and especially to forms of this protein that have been modified by non-enzymatic glycation. The theory and use of both frontal analysis and zonal elution competition studies in such work will be discussed. Various practical aspects of these experi...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Interpenetrating Polymer Network Composite Cryogels with Tailored Porous Morphology and Sorption Properties
Cryogels, by their particular morphology and mechanical properties, proved to be invaluable materials in biomedicine and biotechnology as carriers for molecules and cells, chromatographic materials for cell separations and cell culture. Methods used in the characterization of porosity and sorption properties of cryogels are very needful tools, which assist the investigator in the decision on the performances of the gel. Herein, we describe the preparation of ionic interpenetrating polymer network composite cryogels and the characterization methods of their porous morphology, and then the methods used for testing their sorp...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Molecularly Imprinted Cryogels for Human Serum Albumin Depletion
Molecularly imprinted polymers can be used for the selective capture of a target molecule from complex medium. Cryogels novel matrices, which characterized by their supermacropores that makes their use advantageous when studying with biological samples. By combining high selectivity of the molecular imprinting approach with using cryogel as a base polymer, in this protocol, preparation of the albumin-imprinted cryogels is described. This material is a useful candidate for the selective albumin depletion from the human serum sample prior to the detailed proteomic analysis. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
A Novel Chromatographic Media: Histidine-Containing Composite Cryogels for HIgG Separation from Human Serum
Histidine-containing microspheres (HCM) with 2 μm in size were synthesized by suspension polymerization of poly(hydroxyethyl methacrylate) and N-methacryloyl-l-histidine methyl ester. Then, they were used to prepare composite cryogel columns by an embedding process for affinity depletion of immunoglobulin G (HIgG) from human serum via histidine groups on microspheres. Here, we describe HIgG adsorption performance of composite cryogel columns in both aqueous solution and human serum. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Ni(II) Chelated IDA Functionalized Poly(HEMA-GMA) Cryogels for Urease Adsorption
Cryogelic support materials have been intensively used for the purification and separation of biomolecules. Cryogels are cheap materials, they can be easily used for different purposes and their chemical and physical stabilities are very high. Cryogels can also be easily functionalized with different type of ligands and are be applicable to different affinity systems. Within these affinity systems, immobilized metal affinity chromatography (IMAC) offers efficient and simple protein purification strategies. IMAC technology has been deeply applied to bioseparations studies. In the present chapter, the preparation of a cryoge...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography
Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90 %. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gra...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Cryogels with Affinity Ligands as Tools in Protein Purification
Affinity chromatography is one of the well-known separation techniques especially if high purity is desired. Introducing ligands on monolithic structure gives the possibility for purifying complex media such as plasma and crude extract. This chapter is focusing on the preparation of cryogels as monolithic column and immobilization of concanavalin A on its surface as ligand for capturing the glycoprotein horseradish peroxidase. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
Introduction to Macroporous Cryogels
Cryogels are highly elastic three-dimensional materials consisting of a network of interconnected macropores. This unique morphology combined with high mechanical and chemical stability provides excellent mass flow properties. The matrices are synthesized at subzero temperatures from almost any gel-forming precursor. The main fields of application are in biotechnology as 3D-scaffold for cell cultivation, and tissue engineering, or bioseparation as chromatographic media for the separation and purification of biomolecules. This chapter briefly highlights the preparation, properties, and application of these materials. (Sourc...
Source: Springer protocols feed by Biochemistry - March 9, 2015 Category: Biochemistry Source Type: news
