Television-based densitometric analysis of proteins separated by two-dimensional gel electrophoresis

Aycock, B.F.; Weil, David E.; Sinicropi, Dominick; McIlwain, D. L.

doi:10.1016/0010-4809(81)90003-3

Cited by 25 publications

(2 citation statements)

References 9 publications

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“…The integrated (total) optical density (IOD) of individual stained proteins in twodimensional gels was measured with the television-based system described previously (2). The 1OD of each spot was divided by the IOD of ovalbumin, a fixed amount of which was added as an internal standard to each sample prior to electrophoresis, to normalize for differences in the degree of staining or in the amount of protein that entered each gel.…”

Section: Densitometry Of Two-dimensional Electrophoretic Gelsmentioning

confidence: 99%

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons.

Sinicropi¹,

McIlwain²

1983

The Journal of Cell Biology

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Changes in the amounts of tubulin, actin, and neurofilament polypeptides were found in regenerating motoneurons of grass frogs during the period of axonal elongation. Ventrat roots 9 and 10 were transected unilaterally about 7 mm from the spinal cord. 35 d later, [3H]colchicine binding had decreased in the proximal stumps to approximately one-half of contralateral control values, well before the regenerating motor axons had reinnervated skeletal muscles of the hind limb.[3H]colchicine binding did not change significantly in the operated halves of the 9th and 10th spinal cord segments over a 75-d period. The relative amounts of actin, tubulin, and neurofitament polypeptides in the operated ventral roots were measured by quantitative densitometry of stained two-dimensional electrophoretic gels. AIpha-tubulin, beta-tubulin, and the 68,000 molecular weight subunit of neurofilaments (NF68) decreased within the transected ventral roots to 78%, 57%, and <15% of control values, respectively. The amount of actin increased to 132% of control values within the operated ventral roots, although this change was not statistically significant. Opposite changes were found within motoneuronal cell bodies isolated from the spinal cord. The relative amounts of alpha-tubulin, beta-tubulin and NF68 within axotomized perikarya increased, respectively, to 191%, 146%, and 144% of that in control perikarya isolated from the contralateral side of the spinal cord. Thus, the changes in NF68 and tubulin did not occur uniformly throughout the injured cells. The possible structural and functional consequences of these changes are discussed.Cytoskeletal proteins, including actin, tubulin, and neurofilameat polypeptides, are particularly abundant within axons and play important roles in axonal structure and function (35,36,67). Axons receive a continuous supply of cytoskeletal proteins from their nerve cell bodies by slow axonal transport (20). When an axon in the peripheral nervous system is transected (axotomy), the isolated distal segment degenerates and is regenerated from the proximal segment (23). Whether or not the normal continuous supply of cytoskeletal proteins from the perikaryon is sufficient for axonal regeneration is unclear at present (21, 30). A prevailing hypothesis states that the synthetic priorities of axotomized neurons become reordered to favor the increased production and delivery of structural proteins to the regenerating axons (19, 49).Altered axonal amounts of cytoskeletal proteins can arise from changes in their synthesis, degradation, and/or transport. Previous studies of cytoskeletal proteins within regenerating neurons have focused on changes in their synthesis and transport. Several laboratories have reported increased rates of tubulin and actin synthesis in the region of axotomized retinal ganglion cell bodies (6,17,26), although such increases were not statistically significant in autonomic and sensory ganglia (24,46). Increased amounts of radioactivity associated with newly synthesized tubulin and actin...

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Section: Densitometry Of Two-dimensional Electrophoretic Gelsmentioning

confidence: 99%

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons.

Sinicropi¹,

McIlwain²

1983

The Journal of Cell Biology

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“…Because a wealth of useful information can be extracted from these gels by simple hand measurements, augmented if necessary with lane-by-lane scanning densitometry, there has been little emphasis on automating the analysis of these gels. This situation contrasts with that for two-dimensional protein gels, in which case the high information content of typical individual gels has provided strong motivation for computerized analysis (1)(2)(3)(4)(5).…”

Section: Introductionmentioning

confidence: 99%

Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments

1984

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A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments.

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Computers in Biochemical Analysis

Crabbe

1985

Methods of Biochemical Analysis

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Television-based densitometric analysis of proteins separated by two-dimensional gel electrophoresis

Cited by 25 publications

References 9 publications

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons.

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons.

Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments

Computers in Biochemical Analysis

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