Malcolm G. Pluskal

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry.

In‐gel peptide digestion has become a widely used technique for characterizing proteins resolved by two‐dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96‐well plate with a low peptide binding membrane and ZipTip® micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.

Deadend Microfiltration: Applications, Design, and Cost

The previous three chapters covered a general description of microfiltration and the theories of deadend and crossflow microfiltration. This chapter focuses on the practical aspects of microfiltration with special emphasis on deadend microfiltration using commercial membranes, important applications, design criteria, and cost estimates.

Membrane surface for carbohydrate analysis

Abstract A poly(methyldiallylamine-chlorohydrin) polymer was used to surface modify microporous PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene), and UPE (ultra high molecular weight polyethylene) membranes. These membranes were used to covalently bond hydrophilic charged carbohydrate molecules. The surfaces were characterized in terms of the structural variations of the monomer units. Immobilization occurs by a two-step process whereby strong imagewise electrostatic forces bring nucleophile (charged carbohydrate conjugate) and electrophile (membrane surface desorptive media groups) into close proximity for a pH triggered reaction to covalently bond the carbohydrate. The immobilization potential of the surfaces is described by the chlorohydrin and epoxy groups available for reaction, and is inversely proportional to the amount of alkali employed during membrane surface modification. Conjugation of complex carbohydrate mixtures to a charged fluorophore, followed by electrophoresis, transfer t...

High-performance ion-exchange chromatography of myosin using a DEAE-5PW column

High-performance ion-exchange chromatography of myosin using a DEAE-5PW packing was used to purify myosin from skeletal, cardiac and smooth muscle. This method produces high-speed resolution (30-min analysis) of myosin from contaminating myofibrillar proteins. The column has a high capacity for binding myosin (up to 1 g) and can be used for small-scale preparation of highly purified myosin. Gel analysis in the presence of sodium dodecyl sulfate showed recovery of myosin with very little contamination of other myofibrillar proteins. Myosin was also recovered from small biopsy samples (0.1 g) by a direct extraction technique with recovery of biological ATPase activity.