The Preparation and Enzymatic Hydrolysis of Reduced and S-Carboxymethylated Proteins

Crestfield, Arthur M.; Moore, Stanford; Stein, William H.

doi:10.1016/s0021-9258(18)81308-4

Cited by 2,607 publications

(324 citation statements)

References 29 publications

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“…NaBH 4 has proteolytic properties, 41 acting on peptide bonds mildly and selectively, 42,43 particularly for serine, threonine, and asparagine, but not others. NaBH 4 can cleave peptide linkages and reduce disulfide bonds.…”

Section: Resultsmentioning

confidence: 99%

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections

Bolognesi

Manzoni

Scalia

et al. 2017

J Histochem Cytochem.

139

146

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Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

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Section: Resultsmentioning

confidence: 99%

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections

Bolognesi

Manzoni

Scalia

et al. 2017

J Histochem Cytochem.

139

146

View full text Add to dashboard Cite

show abstract

“…Samples (0.5-1.Omg) of component LsIlI and reduced and S-carboxymethylated component LsIII (Crestfield et al, 1963;Maeda & Tamiya, 1974) were hydrolysed with 6M-HCI (0.4ml) in sealed glass tubes in vacuo at 105°C for 24, 48 and 72h. The analysis was performed on an automatic amino acid analyser (type JLC-5AH; Japan Electron Optics Laboratory Co., Tokyo, Japan).…”

Section: Amino Acid Analysismentioning

confidence: 99%

The isolation of an easily reversible postsynaptic toxin from the venom of a sea snake, Laticauda semifasciata

Maeda

Takagi

Tamiya

et al. 1974

Biochemical Journal

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A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10-20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate-polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD(50) of component LsIII by intramuscular injection was 1.24mug/g body wt. for mice and 0.45mug/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.

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“…Antigens were used at a concentration of 0.2mg/ml in 0.1M-sodium phosphate buffer. Carboxymethylation ofrabbit transferrin Rabbit transferrin was reduced and carboxymethylated as described by Crestfield et al (1963). Amino acid analysis Protein in sealed evacuated tubes was hydrolysed in either 6 M-HCI/0.…”

Section: Immunological Methodsmentioning

confidence: 99%

The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin

Heaphy¹,

Williams²

1982

Biochemical Journal

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Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain.

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The Preparation and Enzymatic Hydrolysis of Reduced and S-Carboxymethylated Proteins

Cited by 2,607 publications

References 29 publications

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections

The isolation of an easily reversible postsynaptic toxin from the venom of a sea snake, Laticauda semifasciata

The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin

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