Immunodetection of insulin after transfer from gels to nitrocellulose filters. A method of analysis in tissue extracts

Kakita, Keiji; O'Connell, K.; Permutt, M. A.

doi:10.2337/diabetes.31.7.648

Cited by 21 publications

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“…However, it may not always be feasible to control for this artifact using histochemical arguments. In such instances it may be worth using biochemical techniques to explore these questions (Schueler et al, 1986;Kakita et al, 1982).…”

Section: Characterization Of Three-color Immunofluorescencementioning

confidence: 98%

A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

Wessendorf¹,

Appel²,

Molitor³

et al. 1990

J Histochem Cytochem.

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Coexistence of neurotransmitters within single nerve fibers or terminals can be convincingly demonstrated by the use of multicolor immunofluorescence. The present study examined whether three-color immunocytochemical localization of coexisting neurotransmitters can be performed using the blue fluorophore AMCA. Spectrofluorometric examination of secondary antibodies conjugated with AMCA, fluorescein, and lissamine rhodamine showed that the peaks of excitation and emission were well separated and that dots of AMCA-conjugated IgG dried on slides were not visible when viewed using microscope filters for rhodamine and fluorescein. These findings suggest that AMCA might be suitable for three-color immunofluorescence. The usefulness of AMCA for triple labeling was tested directly by staining sections of rat brainstem and spinal cord for serotonin (5HT), substance P (SP), and either enkephalin (ENK) or prepro-thyrotropin-releasing hormone 160-169 (ppT), a marker peptide for thyrotropin-releasing hormone. Triple labeling for 5HT, SP, and ppT was observed in both brainstem and spinal cord but was only very rarely observed for 5HT,SP, and ENK. No evidence was found for artifactual triple labeling, although false negatives appeared to be possible in some circumstances. We conclude that AMCA can be combined with fluorescein and lissamine rhodamine for three-color immunofluorescent studies of coexisting neurotransmitters. In addition, the coexistence of 5HT with ENK appears to be much less common than the coexistence of 5HT with either SP or ppT.

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Section: Characterization Of Three-color Immunofluorescencementioning

confidence: 98%

A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

Wessendorf¹,

Appel²,

Molitor³

et al. 1990

J Histochem Cytochem.

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“…16 After polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose filters, photoaffinity crosslinked, treated with anti-insulin serum, and the immune complexes detected with 125 l-protein A. The amount of 1 2 5 Iprotein A bound has been shown to be proportional to the amount of insulin electrophoresed, 16 so that densitometric tracings of autoradiographs from multiple samples could be obtained to determine the relative proportions of the two insulins. An autoradiograph of insulins I and II in pancreatic extracts from four different mouse strains is illustrated in Figure 1.…”

Section: Pancreatic Content Of Insulins I and Ii In Various Inbred Stmentioning

confidence: 99%

“…16 Transfer of insulin from polyacrylamide gels to nitrocellulose filters by electroblotting was shown to yield quantitative recovery on multiple determinations. The insulin was then covalently attached to the filters by photoaffinity cross-linking with hydroxysuccinimidyl-P-azidobenzoate (HSAB).…”

Section: Animalsmentioning

confidence: 99%

“…The proportions of insulins I and II were determined by an immunoelectrophoretic method as described. 16 The method essentially involves electrophoresis of pancreas extracts in 12.5% polyacrylamide gels (tris-glycine buffer, pH 8.9), transfer to nitrocellulose filters, and detection with antiserum. Autoradiographs of the 125 l-protein A bound to anti-insulin antibody were quantitated by densitometric analysis.…”

Section: Animalsmentioning

confidence: 99%

“…2 Similar proportions were found when insulin biosynthesis was measured in isolated rat islets. 2 -13 " 15 Recently in this lab a rapid immunoelectrophoretic method for analysis of insulin in pancreatic extracts that readily separates insulins I and II 16 has been developed. Growth hormone tumor-bearing hyperinsulinemic rats were found to contain 5-6 times more insulin I than II.…”

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confidence: 99%

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Pancreatic Content of Insulins I and II in Laboratory Rodents: Analysis by Immunoelectrophoresis

Kakita¹,

O’Connell²,

Permutt³

1982

Diabetes

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The purpose of these experiments was to determine the relative proportion of insulins I and II in the pancreas of inbred strains of mice and rats, and whether this proportion would be altered by conditions known to affect insulin biosynthesis. Rapid analysis of multiple samples was accomplished by an immunoelectrophoretic method in which insulin can be detected in crude pancreatic extracts without further purification. Insulin I was found to account for 65-76% of total pancreatic content in C3H/HeJ, C57BL/6J, and CD 2 F 1 mice. Glucose injection produced a significant increase in insulin content, which was completely accounted for by insulin I, although fasting for 2 days had no effect. In all three strains there was either no change or a decrease in content of insulin II, while insulin I increased 77-83% (P < 0.05) of the total pancreatic insulin.To determine the effects of chronic hyperglycemia, the content of insulins I and II was measured in the pancreas of obese hyperglycemic mice and compared with that of lean littermates. Insulin content in pregnant rats was also evaluated, since pregnancy is characterized by insulin resistance and hyperinsulinemia. The results of these studies indicate that insulin I predominates in the pancreas of a variety of strains of rats and mice. More important is the observation that conditions that increase insulin production preferentially increase insulin I relative to II, suggesting independent regulation of the two genes. DIABETES 31:841-845, October 1982.T wo different insulins are present in the pancreas of the rat, 1 -2 mouse, 3 -4 and certain fish, 5 -6 which are encoded by nonallelic genes. 7 -8 The two rat insulin genes are separated by at least 9000 base pairs of DNA, and are 93% homologous in the coding regions. The initial translation products, preproinsulins I and II, differ by only three amino acids in the pre-region, two in the B-chain, and two in the C-peptide. Insulins I and II of the mouse have amino acid compositions identical with those of the rat. 3 -4 The study of diabetes in laboratory rodents has been important because of the obvious advantages for manipulating genetic and environmental conditions. Spontaneous diabetes has been described in at least 10 different strains of laboratory rodents. 9 -10 Because of the relative ease of isolation of pancreatic islets from rodents, insulin biosynthesis and secretion have been extensively investigated in these animals. 11 " 14 In spite of numerous studies, very little is known about the relative expression of the two insulin genes. An often-quoted figure for the proportions of insulins I and II is derived from the observation that crystalline insulin from Sprague-Dawley rats contains approximately 60% insulin I and 40% insulin II. 2 Similar proportions were found when insulin biosynthesis was measured in isolated rat islets. 2 -13 " 15 Recently in this lab a rapid immunoelectrophoretic method for analysis of insulin in pancreatic extracts that readily separates insulins I and II 16 has been developed. Growth h...

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Immunochemical methods

Ferencík

1993

Handbook of Immunochemistry

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Immunodetection of insulin after transfer from gels to nitrocellulose filters. A method of analysis in tissue extracts

Cited by 21 publications

References 0 publications

A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

Pancreatic Content of Insulins I and II in Laboratory Rodents: Analysis by Immunoelectrophoresis

Immunochemical methods

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