Preparation of inside-out vesicles of pig lymphocyte plasma membrane

Walsh, Frank S.; Barber, Brian H.; Crumpton, Michaël J.

doi:10.1021/bi00661a025

Cited by 69 publications

(22 citation statements)

References 32 publications

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“…In our case, the clumping of some intramembranous particles made the use of this method hazardous. Lectin columns have been used by Walsh and co-workers, but they do not allow a separation of plasma membrane "inside-out" vesicles from Golgi vesicles, the presence of which was not searched for (46). In our case, we have demonstrated that plasma membranes and Golgi vesicles co-purify to some extent.…”

Section: Monneron and D'alay~r Enzymatic Composition And Uhrastructurmentioning

confidence: 56%

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

Monneron¹,

d’Alayer²

1978

The Journal of Cell Biology

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The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, 7-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase.Plasma membranes and light nuclear membranes appeared as pure unitmembrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.KEY WORDS lymphocyte plasma membrane nuclear membrane membrane marker enzymes ultrastructure subcellular fractionation T cells play a central role in immunological processes. They are responsible for the recognition of many antigens and respond to them by activating surface signals and releasing soluble factors which trigger B cells in various ways (7,30). They are also the effectors of cellular immunity. Such functions are mediated by the plasma membrane. Work is in progress in several laboratories to characterize the receptors of antigens, now known as the products of the Ir genes, and the surface signals, presumably related to the molecules responsible for histocompatibility (38). To study such molecules, it appears likely that they must be maintained in their structural context. Therefore, it is essential to obtain purified plasma membranes of T cells, and of their precursors, the thymocytes.T and B cells, despite their profound biological differences, are morphologically indistinguishable (1, 5). Thymocytes, the precursors of T cells, resemble very much circulating lymphocytes when isolated in a culture medium. Therefore, methods J. C~LL BIOLOgy 9 The Rockefeller University Press 9 0021-9525/78

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Section: Monneron and D'alay~r Enzymatic Composition And Uhrastructurmentioning

confidence: 56%

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

Monneron¹,

d’Alayer²

1978

The Journal of Cell Biology

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“…Although efficient lymphocyte extraction from whole blood is well established, there is a certain amount of residual protein contamination due to immunoglobulins, albumin and other abundant proteins. (30–32)…”

Section: Resultsmentioning

confidence: 99%

Improved data normalization methods for reverse phase protein microarray analysis of complex biological samples

Chiechi¹,

Mueller²,

Boehm³

et al. 2012

Biotech

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Reverse phase protein microarrays (RPMA) are designed for quantitative, multiplexed analysis of proteins, and their posttranslational modified forms, from a limited amount of sample. To correct for sample to sample variability due to the number of cells in each lysate and the presence of extracellular proteins or red blood cells, a normalization method is required that is independent of these potentially confounding parameters. We adopted a gene microarray algorithm for use with RPMA to optimize the proteomic data normalization process and developed a systematic approach to RPMA processing and analysis, tailored to the study set. Our approach capitalizes on the gene microarray algorithms geNorm and NormFinder to identify the normalization parameter with the lowest variability across a proteomic sample set. Seven analytes (ssDNA, glyceraldehyde 3-phosphate dehydrogenase, α/β-tubulin, mitochondrial ribosomal protein L11, ribosomal protein L13a, β-actin, and total protein) were compared across sample sets including cell lines, tissues subjected to laser capture microdissection, and blood-contaminated tissues. We examined normalization parameters to correct for red blood cell content. We show that single-stranded DNA (ssDNA) is proportional to total non-red blood cell content and is a suitable RPMA normalization parameter. Simple modifications to RPMA processing allow flexibility in using ssDNA- or protein-based normalization molecules.

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“…polypeptide was immunoprecipitated from the 1251-labelled plasma membrane vesicles ( Figure 3, track 4). Since the plasma membrane preparation most probably comprises vesicles of mixed orientation (Walsh et al, 1976), polypeptides located on either the cell surface or cytoplasmic side of the membrane will be labelled. The immunoprecipitation data confirm the results of the immunofluorescence experiments and indicate that p68 is located intracellularly in lymphocytes.…”

Section: Immunoprecipitation From Lymphocytesmentioning

confidence: 99%

Cellular distribution of p68, a new calcium-binding protein from lymphocytes.

Owens¹,

Gallagher²,

Crumpton³

1984

The EMBO Journal

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A Ca2+‐binding protein of mol. wt. 68 000 (p68) is a major component of a Nonidet P‐40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity‐purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically‐ but not surface‐labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane‐bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub‐fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non‐lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent‐extracted cells the protein was localised in a lamina‐like network. A similar immunofluorescent staining pattern has recently been observed for spectrin‐related proteins in the detergent‐resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub‐membranous cytoskeletal complex not only in lymphocytes but also in other cell types.

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Preparation of inside-out vesicles of pig lymphocyte plasma membrane

Cited by 69 publications

References 32 publications

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

Improved data normalization methods for reverse phase protein microarray analysis of complex biological samples

Cellular distribution of p68, a new calcium-binding protein from lymphocytes.

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