Aldo M. Pitt

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.

High Throughput Screening Protein Kinase Assays Optimized for Reaction, Binding, and Detection Totally within a 96-Well Plate

Signal transduction assays, particularly for protein kinases, are an area of increasing interest and activity for laboratories investigating the regulation of cellular functions. The traditional kinase assay methods require the tedious and time consuming manipulation of phosphocellulose disks typically used to bind the phosphorylated substrate. Drug discovery research requires the availability of rapid and reliable procedures to evaluate large numbers of samples for bioactivity. The 96-well phosphocellulose MultiScreen(r) assay plates were specifically developed to meet these assay requirements. A cyclic-AMP-dependent protein kinase A (PKA) assay was optimized to be performed entirely within the phosphocellulose MultiScreen plate, including reagent and sample addition, incubation, washing, and direct microplate scintillation counting. The protocol is directly adaptable to a high throughput kinase screen. Both the Kemptide peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) and the histone H1 protein were used as the phosphorylation substrates. Crude and purified PKA enzymes were found to have a sensitivity of 0.4 U for Kemptide substrate, which was comparable to the assay performed by the traditional transfer to phosphocellulose paper. The results demonstrate that kinase assays can be performed entirely in a MultiScreen phosphocellulose plate.

Microporous membrane growth substrates for embryonic stem cell culture and differentiation.

As the field of embryonic stem cell culture and differentiation advances, many diverse culturing techniques will ultimately be necessary in order to fully reproduce the various environments these cells normally encounter during development. Although most of the work to date has been performed on solid plastic supports, this growth support has several limitations in its representation of the in vivo environment. Impermeable substrates force the cells to exchange their gas and nutrients exclusively through the top side of the cultured cells. In contrast, cells growing in vivo are exposed from several directions to factors from the blood, other cells, soluble factors, and liquid-air interfaces. Additionally, solid plastic presents a smooth two-dimensional surface that is not experienced in vivo. Therefore, the use of traditional plastic presents limitations upon normal cellular morphology, function, and differentiation. An important alternative to growth on solid plastic is the growth of cells on microporous membranes. One of the many advantages to cell growth on porous membrane substrates is their ability to provide a surface that better mimics a three-dimensional in vivo setting. A porous membrane allows multidirectional exposure to nutrients and waste products. In addition, the membrane separation of dual chambers allows for the coculture of cells of different origin to study how cells interact through indirect signaling or through providing a conditioned niche for the proper growth and differentiation of cell types.

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.

Sample Preparation for High Throughput Screening of Complex Biomolecules Using a Multidimensional Approach

5000 4000 4 00 Mass (mIz) 300 HPLC and/or MALDI-TOF. Materials and Methods: 96-well min i-chromatography columns were prepared following the manufacturers instructions by loading dry media into MultiScreen Resist O.4mm filter plates (MAR4N0410 , Millipore Corporation , Bedford MA) using eithe r a 25pl or a 45pl MultiScreen Column Loader (Millipore Corporation , Bedford MA). Media used included reverse phase resins of a) 300 A6-10 pm C18 (Waters-Microbondapak, Waters Corporation, Milford, MA); b) 300 A l Surn or 40 pm C4 (Amicon-Matrex, Millipore Corporation, Bedford, MA)j or c) R2 styrene-divinylbenzene(DVB), C4 like (PE-PerSeptive Biosystems,