Showing papers by "Paul Matsudaira published in 1989"

A Practical guide to protein and peptide purification for microsequencing

Abstract: Introduction. Strategies for Obtaining Partial Amino Acid Sequence Data from Small Quantities (5nmol) of Pure of Partially Purified Protein. Enzymatic Digestion of Proteins and HPLC Peptide Isolation. Purification of Proteins and Peptides By SDS-PAGE. Internal Amino Acid Sequence Analysis of Proteins After in Situ Protease Digestion on Nitrocellulose. Mass Spectrometric Strategies for the Structural Characterization of Proteins. Appendix. References. General Reference Books on Protein Microsequencing Methodologies.

Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium.

Abstract: The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8.5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2–3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2–3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.

Initial and Repetitive Yields from Proteins Blotted on PVDF Membranes

Abstract: PVDF (polyvinylidene difluoride) membranes have proven to be a useful support on which proteins or peptides can be immobilized during automated Edman degradation procedures (Matsudaira 1987). Several groups have reported that the initial and repetitive yields for samples electroblotted onto PVDF membranes (Baum et al 1987; Legendre and Matsudaira 1988; Moos et al 1988; Xu et al 1988; Yuen et al 1988) are much higher than samples blotted onto activated glass fiber filters (Vandekerchove et al 1985; Aebersold et al 1986). Since most of the measurements involved 100–500 pmole quantities of material, it would be valuable to evaluate the yields of < 100 pmole quantity of sample. In this study, I measured the initial and repetitive yields of 4–110 pmole quantities of 125I-s-lactoglobulin. The data suggests that both the repetitive and initial yields are independent of sample quantity and that sample loss during the sequencing cycles significantly affects the repetitive yields. Based on these results, it appears that sample detection is the major limit to routine sequence analysis of subpmole quantities of material.