An improved scintillation cocktail of high-solubilizing power

Anderson, Larry E.; McClure, William O.

doi:10.1016/0003-2697(73)90465-x

Cited by 412 publications

(76 citation statements)

References 13 publications

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“…3C. The same level of binding was achieved after [10][11][12] [10][11][12][13][14][15][16][17][18][19][20] molecules per cell. Although this is in good agreement with other estimates of the binding capacity of disc cytosol, it is much lower than the binding capacity of whole discs-500-1000 20-hydroxyecdysone sites per cell (9) and 6001000 PNA sites per cell (unpublished data)-and represents only 2-4% of the total ecdysteroid-specific binding capacity of the cell.…”

mentioning

confidence: 77%

“…The high level of nonspecific binding at physiological concentrations of 20-hydroxyecdysone (0.1,uM), the high equilibrium Kd, and the low specific activity of available 20-hydroxyecdysone placed significant technical constraints on further work. Ponasterone A (PNA), an ecdysteroid approximately 50 times more active than the native hormone (9), has been shown to be identical to 20-hydroxyecdysone in its action on discs, differing only in the effective concentration (ref. 9; unpublished data).…”

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confidence: 99%

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Ecdysteroid receptors in imaginal discs of Drosophila melanogaster.

Yund¹,

King²,

Fristrom³

1978

Proc. Natl. Acad. Sci. U.S.A.

106

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confidence: 77%

mentioning

confidence: 99%

Ecdysteroid receptors in imaginal discs of Drosophila melanogaster.

Yund¹,

King²,

Fristrom³

1978

Proc. Natl. Acad. Sci. U.S.A.

106

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“…The gels were then removed, stained with 0.1% Coomassie Brilliant Blue, and destained (13). The gel was sectioned into 2-mm slices, each of which was oxidized in an individual vial with periodic acid (12) and counted after the addition of Triton-xylene scintillation fluid (14).…”

Section: Methodsmentioning

confidence: 99%

Differential Transport of Protein in Axons: Comparison Between the Sciatic Nerve and Dorsal Columns of Cats

Anderson

McClure

1973

Proc. Natl. Acad. Sci. U.S.A.

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Fast axoplasmic transport was studied in dorsal root ganglion cells of the cat. Proteins carried in the fast axoplasmic flow were labeled after an intraganglionic injection of L-[4,5-3Hlleucine. The rate of transport was 380 + 26 mm/day in both the central and peripheral branches of the bifurcating axons that arise from cells of the dorsal root ganglion. The amount of radioactivity transported centrally, through the dorsal roots into the spinal cord, was about 50% of that moving peripherally, through the sensory fibers of the sciatic nerve. Labeled material appears to be transported principally in a bound form, as 70-80% of the radioactivity was insoluble in 0.01 M potassium phosphate buffer at pH 7.4. With multiple extractions, a fractionation procedure was developed by which 94-96% of the total transported radioactivity could be solubilized. The proteins carried by fast axoplasmic transport through the dorsal columns and through the sciatic nerve were compared by electrophoresis of extracted fractions on Na dodecyl sulfate-polyacrylamide gels.Different patterns of radioactivity are seen in the electrophoretograms, suggesting that the cells of the dorsal root ganglion may possess the ability to commit different proteins to transport through two branches of a single bifurcating axon.Neurons elaborate various protein and particulate matter that is carried through the axon to the nerve terminals by fast axoplasmic transport (1, 2). This phenomenon has been demonstrated in sensory fibers of the sciatic nerve after injection of [3HIleucine into the seventh lumbar dorsal root ganglia (Lq DRG) of cats (3, 4). Labeled material also moves from the DRG through the dorsal root fibers into the central nervous system (3). Recent work has confirmed the importance of fast transport in the control and maintenance of synaptic function (5, 6).In this paper, we consider the question: Can different proteins be transported through different branches of a bifurcating axon? The cells of the Lq DRG in cats provide a useful experimental system. Each of these cells sends out a single bifurcating fiber (7). From the bifurcation, one branch extends toward the periphery as a sensory fiber in the sciatic nerve. The other branch travels through the dorsal roots and into the spinal cord, where the fiber either terminates within a short distance or extends up the dorsal columns to the dorsal column nuclei in the medulla. In this study, labeling with radioactive precursors was used to compare material transported through the peripheral fibers, in the sciatic nerve, with that transported through the central fibers, in the dorsal columns. Although the rate of movement and the general Abbreviations: DRG, dorsal root ganglia; L7, lumbar seventh. 1521 solubility characteristics of the labeled material were the same for material moving in both directions, different transported proteins were found in the two branches. MATERIALS AND METHODSCats (between 2.5 and 4.0 kg) were anesthetized by intraperitoneal injection of sodium pentobarbital (aver...

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“…The assay differed from the standard one in that 7/lCi of [3HjUTP was substituted for [1X_ 32 PjUTP. Counting was performed in Triton X-114/xylene-based scintillation fluid at 25 % efficiency (Anderson and McClure 1973).…”

Section: Determination Of Chain Lengthsmentioning

confidence: 99%

Characterization of the RNA Synthesizing Activity of Isolated Kidney Nuclei

Cj¹,

Jf²

1975

Aust. Jnl. Of Bio. Sci.

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The limiting factor in RNA synthesis by isolated kidney nuclei is RNA nucleotidyltransferase at high salt concentrations but at low salt concentrations template availability becomes limiting.oc-Amanitin inhibits 85 % of the activity at high salt concentrations but only 20-50 % of the activity at low salt concentrations. Exogenous DNA is utilized at low salt concentrations [up to O·lM (NH4hS041 but not at high salt concentrations. The effect of increasing salt concentration is mainly to cause an increase in the length of chains synthesized. Initiation rates are not increased by high salt concentrations. The apparent Km for UTP is 8-10 11M at high salt concentrations, indicating that assays performed at low UTP concentrations are likely to give inaccurate results. The activation energy for the reaction at low salt concentration is less than that for the reaction at high salt concentration. The RNA synthesizing capacity of kidney nuclei is dependent on the method of isolation, and preparation by a modification of the Chauveau method (Chauveau et al. 1956) yields the most active nuclei.

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An improved scintillation cocktail of high-solubilizing power

Cited by 412 publications

References 13 publications

Ecdysteroid receptors in imaginal discs of Drosophila melanogaster.

Ecdysteroid receptors in imaginal discs of Drosophila melanogaster.

Differential Transport of Protein in Axons: Comparison Between the Sciatic Nerve and Dorsal Columns of Cats

Characterization of the RNA Synthesizing Activity of Isolated Kidney Nuclei

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