High resolution two‐dimensional polyacrylamide gel electrophoresis. II. Analysis and applications

Dünn, Michael J.; Burghes, Arthur H.M.

doi:10.1002/elps.1150040302

Cited by 55 publications

(13 citation statements)

References 167 publications

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“…6D), they were completely absent from the stained gel. Because silver staining is 100 to 200 times more sensitive than Coomassie blue staining (13), the results of this experiment suggest that the hsp are synthesized in very low amounts or turn over quickly. We cannot eliminate the possibility that the hsp do not bind silver efficiently (29), although it seems unlikely that such a large number of proteins, varying in size as well as charge, would all not bind silver.…”

Section: Methodsmentioning

confidence: 84%

Tissue Specificity of the Heat-Shock Response in Maize

Cooper

Ho²,

Hauptmann³

1984

Plant Physiol.

130

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ABSTRACFThe tissue specificity of the heat-shock response is maize was investigated. The ability to synthesize heat shock proteins (hsp) at 40°C, as well as the intensity and duration of that synthesis, was analyzed in coleoptiles, scutella, green and etiolated leaves, snspensin-cultured cells, germinating pollen grains, and primary root section at different stages of development. One-dimensional sodium dodecyl sulfate gel electrophoresis of extracted proteins revealed that most of the tissues synthesized the typical set of 10 hsp, but that the exact charcterstics of the response depended upon the tissue type. While elongating portions of the primary root exhibited a strong heat shock response, the more mature portions showed a reduced ability to synthesize hsp. Leaves Higher plants have been shown to respond to elevated temperatures by synthesizing a set of hsp3 (1,5,11,18 In this report, the heat-shock response was investigated in a variety of maize tissues, including coleoptiles, scutella, green and etiolated leaves, suspension-cultured cells, germinating pollen gains, and primary roots at different stages of maturity. The ability ofthe tissue to synthesize hsp, as well as the intensity and duration of that synthesis, were analyzed. Also presented is evidence that hsp accumulate only to very low levels in maize. MATERIALS AND METHODSPlant Materials and Incubation Conditions. For studies involving roots, coleoptiles, scutella, and leaves, Crow's Hybrid No. 222 corn (Zea mays L., var A632 x C1042) was used. Roots coleoptiles were obtained from 3-d-old seedlings which had been germinated in darkness at 29°C in gla trays on paper towels kept moist with 0.1 mm CaCl2 solution. Scutella were from seeds imbibed for 24 h under similar conditions.Roots were excised and incubated in the manner previously described (11). Coleoptiles were separated from the rest of the plant, the 1-mm tip removed, and the 1 cm segment immediately below used for the experiment. The segment was longitudinally sliced in half, with one-half used as the control and the other half as the treatment. Half-segments from five plants were combined as a single sample and incubated in the major and minor salts of MS (27) medium as previously described (1 1). Scutella were isolated by aseptically removing the embryos from the seed and then dissecting out the embryonic axis. The scutella were incubated in pairs in a flask containing 2 ml MS salts. The flasks were shaken at 120 cycles/min in a constant temperature bath. The medium was replaced by 2 ml fresh MS salts at the appropriate temperature prior to addition of radioactive label. Unless otherwise stated, roots, coleoptiles, and scutella were incubated at 28°C for 4 h to allow them to overcome the shock of excision prior to any additional temperature treatment. All manipulations involving scutella and coleoptiles occurred in dim green light.For experiments involving leaves, seeds were planted in plastic dishpans in vermiculite and germinated in darkness at 29°C. Once the coleoptiles had emerged thro...

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Section: Methodsmentioning

confidence: 84%

Tissue Specificity of the Heat-Shock Response in Maize

Cooper

Ho²,

Hauptmann³

1984

Plant Physiol.

130

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“…An often quoted report by Poehling and Neuhoff (45) states that "Silver does not stoichiometrically stain proteins, unlike Coomassie Blue". However, their silver-stain data actually is linear over a 30-fold range in protein concentration, while their Coomassie Blue data is only linearity over a 20-fold range (9,36).…”

Section: Quantitation With Silver Stainsmentioning

confidence: 97%

“…However, other reducing agents, such as beta-mercaptoethanol, do not enhance positive image formation. Alternatively, all of the positive image enhancing compounds may simply act as nucleation centers for silver reduction after forming complexes with the proteins (9).…”

Section: Silver Stain Mechanismsmentioning

confidence: 99%

“…The adaptation of the de Olmos histological silver stain as a general protein stain by Merril and Switzer 1979, and the recognition of the large quantitative gain in sensitivity that silver staining offers, over the commonly used organic stain (32,52), has stimulated the recent widespread use of silver staining for the detection proteins separated by polyacrylamide gel electrophoresis. This use of silver has been enhanced by the development of numerous simplified silver stain protocols (9).…”

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confidence: 99%

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Development and Mechanisms of Silver Stains for Electrophoresis

Merril

1986

Acta Histochem. Cytochem

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“…Over the appropriately controlled ranges, integrateddensities ofsilver-stained spots were linearly related to the amount of protein present (25,26). Quantitation of two-dimensional gels requires expensive computer and image analysis hardware and sophisticated software (27 With the combination of the RP, cryomicrodissection, and two-dimensional gel analysis, we can now begin to glean information on the localizations and diffusivities of individual proteins within the living cell. Diffusive proteins equilibrate with the RP, whose water possesses the solvent properties of ordinary water.…”

Section: Analysis Of Protein Content By Two-dimen-mentioning

confidence: 99%

Diffusive and nondiffusive proteins in vivo.

Paine¹

1984

The Journal of Cell Biology

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High resolution two‐dimensional polyacrylamide gel electrophoresis. II. Analysis and applications

Cited by 55 publications

References 167 publications

Tissue Specificity of the Heat-Shock Response in Maize

Tissue Specificity of the Heat-Shock Response in Maize

Development and Mechanisms of Silver Stains for Electrophoresis

Diffusive and nondiffusive proteins in vivo.

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