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M A Mansfield

Publications and source records attributed to M A Mansfield.

7 recordsLinked to original sources
PubMed1999

Immobilon-Ny+ nucleic acid blotting membrane: an advanced nylon membrane optimized for superior fixation and reprobing.

The use of charged nylon membranes in nucleic acid blotting applications has become an important factor in the success of hybridization-based assays. Retention of nucleic acids on these membranes is promoted by baking at 80 degrees C under vacuum or by exposure to short wavelength UV light, with the latter method preferred. Immobilon-Ny+ is an advanced, positively charged nylon membrane that has been optimized to show superior retention of target DNA and RNA under hybridization conditions. Higher signal levels are obtained in these assays compared to competitive membranes, even after 13 cycles of probing. This report illustrates the superior performance of Immobilon-Ny+ in 32P and chemiluminescent hybridization assays on blotted DNA and RNA.

Blotting, Northern↗
PubMed1995

Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking.

The data presented here (Fig. 2, Table 2) clearly demonstrate that wetting of Immobilon-P is not required for immunodetection of transferrin. It can be inferred that the immobilized transferrin undergoes sufficient rehydration at the molecular level to permit epitope recognition by the antibodies, even when the surrounding areas of PVDF remain hydrophobic. The hydrophobic blot procedure was compatible with blots prepared by both tank and semidry transfer and with the substrates BCIP/NBT and 4CN. Additionally, the binding specificity of antitransferrin antiserum was not altered in the hydrophobic blot protocol. Given the diversity of blocking agents, antibodies, and visualization systems available, optimization for specific reagent combinations may be necessary. The most important parameters will be the concentration of blocking agents and detergents used since either, depending on chemical properties and concentration, may cause the membrane to wet out during incubation. This procedure is not applicable to nitrocellulose membranes since surfactants are used in their manufacture specifically to cause wetting in aqueous buffers.

Animals↗
PubMed1988

Cytoplasmic distribution of heat shock proteins in soybean.

Previous analyses of the distribution of heat shock (hs) proteins in soybean (Glycine max L. Merr., var Wayne) have demonstrated that a fraction of the low molecular weight hs protein associates with ribosomes during hs. To more specifically characterize the nature of this association, isokinetic centrifugation of ribosomes through sucrose gradients was used to separate monosomes from polysomes. The present analysis demonstrated that hs proteins were bound to polysomes but not monosomes. Treatment of polysomes with puromycin, K(+), and Mg(2+), which caused dissociation of ribosomes into 40S and 60S subunits, also caused dissociation of the hs proteins. Using the procedure of Nover et al. (1983, Mol. Cell Biol, 3: 1628-1655), a hs granule fraction was also isolated. As in tomato cells, hs granules from soybean seedlings contained the low molecular weight hs proteins as a primary component and a number of other non-hs proteins of relative molecular mass 30 to 40 kilodaltons and 70 to 90 kilodaltons. On metrizamide gradients they exhibited a buoyant density of 1.20 to 1.21 grams per cubic centimeter, typical of ribonucleoprotein particles. Heat shock granules were characterized as unique cytoplasmic particles based on protein composition and buoyant density. Isopycnic centrifugation of ribosome preparations demonstrated that they contained hs granules, but the hs proteins bound to polysomes were not released by KCI/EDTA treatment. Thus, the polysome-bound hs proteins and the granule-bound hs proteins appear to represent two distinct populations of hs proteins in the cytoplasm. Heat shock granules were not distinguishable from ribosomes at the level of resolution used in transmission electron microscopy.

Journal Article↗
PubMed1987

Synthesis of the low molecular weight heat shock proteins in plants.

Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.

Journal Article↗