Publications
-
Description:
Current methods for the separation and purification of RNA are explained, addressing both fundamental principles and practical aspects. The complete know-how to develop and optimize laboratory protocols for the handling of every RNA species, from the very small to the very large. For more information, visit: RNA Purification and Analysis
-
Description:
This separation method is increasingly finding applications in environmental, food, pharmaceutical and clinical analysis. The completely revised and updated fourth edition of this best-selling classic is a thorough treatment of the subject while remaining concise and readable. Alongside ion exchange resins, detectors, ion chromatographic separations, anion, cation and ion exclusive chromatography, special techniques and chemical speciation, new additions include capillary electrophoresis, monolithic columns, zwitterion columns, DNA/RNA analysis, fundamentals of the science of IC, and micro methods. The whole is rounded off by handy tables with details on, among others, detection or elution conditions, making this a ready reference for analytical, environmental and food chemists, chromatographers, pharmaceutists, and chemists working in trace analysis. For more information, visit: Ion Chromatography
-
Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatographyAnalytical Biochemistry, 393 (2009) 8-22
Abstract:
Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.
-
Sample Preparation for the Analysis of Complex Carbohydrates by Multicapillary Gel Electrophoresis with Light-Emitting Diode Induced Fluorescence DetectionAnalytical Chemistry, Vol. 80, No. 11, June, 2008
Abstract:
This paper evaluates various sample preparation methods for multicapillary gel electrophoresis based glycan analysis to support electrokinetic injection. First the removal of excess derivatization reagent is discussed. Although the Sephedex G10 filled multiscreen 96-well filter plate and Sephadex G10 filled pipet tips enabled increased analysis sensitivity, polyamide DPA-6S pipet tips worked particularly well. In this latter case an automated liquid handling system was used to increase purification throughput, necessary to feed the multicapillary electrophoresis unit. Problems associated with the high glucose content of such biological samples as normal human plasma were solved by applying ultrafiltration. Finally, a volatile buffer system was developed for exoglycosidase-based carbohydrate analysis.
-
Boronic acid lectin affinity chromatography (BLAC). 2. Affinity micropartitioning-mediated comparative glycosylation profilingAnal Bioanal Chem, University of Innsbruck, Austria, Epub Rec. 4 May 2008
Abstract:
As a continuation of our work on boronic acid lectin affinity chromatography (BLAC), in this paper we introduce an automated affinity micropartitioning approach using combined boronic acid and concanavalin A (BLAC/Con A) resin-filled micropipette tips to isolate and enrich human serum glycoproteins. The N-linked oligosaccharides of the partitioned glycoproteins were removed by PNGase F enzyme digestion, followed by 8-aminopyrene-1,3,6-trisulfonic acid labeling. Capillary gel electrophoresis with blue LED-induced fluorescence detection was applied in a multiplexed format for comparative glycan profiling. The efficiency of BLAC affinity micropartitioning was compared with that of the individual lectin and pseudolectin affinity enrichment. Finally, we report on our findings in glycosylation differences in human serum samples from healthy and prostate cancer patients by applying BLAC/Con A micropipette tip-based enrichment and comparative multicapillary gel electrophoresis analysis of the released and labeled glycans.
-
Boronic acid-lectin affinity chromatography. 1. Simultaneous glycoprotein binding with selective or combined elutionAnal Bioanal Chem, Vol. 389, October, 2007
Abstract:
We introduce a novel combination of boronic acid affinity chromatography with lectin affinity chromatography, dubbed as boronic acid-lectin affinity chromatography (BLAC). Concanavalin A and wheat germ agglutinin lectins were mixed with the pseudo-lectin boronic acid to form the BLAC affinity column and their performance was evaluated with standard glycoproteins. Optimization of the binding and elution buffers for the BLAC system is described. The BLAC columns were employed to isolate glycoproteins of interest using both selective and/or combined elution.
-
An automated microscale chromatographic purification of virus-like particles as a strategy for process developmentBiotechnology and Applied Biochemistry, Vol. 47 (Part 2), 131-9, June 2007
Abstract:
The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.
-
Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteinsAnalytical Chemistry, Inhibitex, Alpharetta, GA, Rec. 11 April 2007
Abstract:
A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 minutes. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.
-
High-throughput affinity ranking of antibodies using surface plasmon resonance microarraysAnalytical Biochemistry, Vol 351, 241-253, February 2006
Abstract:
A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.
-
Micro-scale open-tube capillary separations of functional proteinsAnalytical Biochemistry, 2006 Mar 1;350(1):128-37. Epub 2006 Jan 17
Abstract:
This article describes a novel technique whereby fully functional proteins or multiprotein complexes are efficiently extracted from biological samples to chemically derivatized walls of fused-silica open-tube capillary columns. Proteins are eluted with very high yields into elution volumes that are smaller in volume than the internal volume of the open-tube capillary column itself, thereby achieving 100-fold increases in target protein concentrations from starting samples of less than 1 ml. The open-tube capillary columns are designed for single use; combined with the physical and chemical characteristics of the open-tube capillary column, this provides exceptional purity to the eluted proteins. Affinity-based open-tube capillary columns are demonstrated here to purify, enrich, and maintain functionality for a monomeric and dimeric enzyme, a low-abundance HeLa nuclear complex, and a light-harvesting octadecameric membrane protein complex. The design of the open-tube capillary column allows for facile direction of the processed protein sample to any number of final detection techniques and is capable of generating final protein concentrations required for many structural biology experiments. The open-tube capillary columns are also characterized by exceptional ease of use. Current designs allow for up to 10 open-tube capillary columns to be applied simultaneously with no fundamental impediments to even greater parallel operation.
-
Protein purification: pure but not simpleNature, Vol 434, 795-798, April 7, 2005
Protein Purification, Technology Feature
Pure but not simpleSmall-Scale Separation
Excerpt:
Sometimes you can do more with less. Proprietary pipette tips developed by PhyNexus in San Jose, California, promise high performance in tiny volumes with minimum fuss. The key lies in encapsulating very small quantities - just 5-10 microlitres - of protein separation resin between hydrophilic screens in the very end of the pipette tip.
-
Striving for purity: advances in protein purificationNature, Vol. 2, No. 1, 71-76, January 2005
Technology Feature
Striving for purity: advances in protein purification
Excerpt:
A substantial bottleneck in working with proteins, both native and recombinant, is purifying the protein of interest efficiently, with a minimum of labor and cost. Recent advances in purification technology from many companies are making the protein scientist's job easier. Caitlin Smith reports.
-
Drug discovery - antibody enrichment and characterizationGenetic Engineering News, Vol. 25, No. 1, January 2005
Genetic Engineering News
Drug Discovery
Antibody Enrichment and CharacterizationTutorial: Utilizing the PhyTip Sample Preparation System
Excerpt:
The rapidly growing field of antibody engineering continues to create the demand for technologies that increase the productivity and throughput of protein interaction analysis. An interest in characterizing protein interactions has increased, so has the number of high throughput analytical technologies (microarrays, protein chips, mass spec, etc.) that offer the ability to study large numbers of samples in fine detail. However, as proteins are inherently complex molecules, elucidation of protein interactions requires that the samples for study are of sufficient purity and activity to produce reliable, high-quality data.