Microisolation of DABITC-derivatized protein by gel electrophoresis: Application to the purification of antibody heavy and light chains

Aebersold, Ruedi; Ledermann, Fritz; Braun, Dietmar; Chang, Jui‐Yoa

doi:10.1016/0003-2697(84)90245-8

Cited by 4 publications

(3 citation statements)

References 13 publications

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“…Amino acid composition was determined by the DABS-CI method [I 51. SDS-gel electrophoresis of DABITCderivatized proteins and peptides were performed as described [18]. Amino-terminal amino acids were quantitatively determined by the DABITC method [I61 with two slight modifications: (a) 5 pl of phenylisothiocyanate was added after DABlTC coupling, and coupling was allowed to proceed for another 15 min at 70 'C before heptaneiethyl acetate extraction, and (b) 4 M HCl/acetic acid (1 : 2, v/v) was used for the cleavage reaction.…”

Section: A V~t Q~s a L T T~p G E T V~l T C~s S T G A V T T S F~y A mentioning

confidence: 99%

Thrombin specificity

Chang

Alkan

Hilschmann

et al. 1985

European Journal of Biochemistry

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Selective cleavage of polypeptides by a-thrombin can be reasonably predicted [Chang, J.-Y. (1985) Eur. J . Biochem. 151,2241. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse K light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArgThr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by a-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9 -10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1 -96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) 1 light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108 -11 1) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human 1 chain (KERN, NEI, NEW, VOR) and one mouse A chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human i chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3 -4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20.

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Section: A V~t Q~s a L T T~p G E T V~l T C~s S T G A V T T S F~y A mentioning

confidence: 99%

Thrombin specificity

Chang

Alkan

Hilschmann

et al. 1985

European Journal of Biochemistry

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“…2 To prepare prestained standards, purified proteins can be covalently modified by various dyes, generating protein derivatives carrying different colors. [3][4][5][6][7] In this manner, a series of prestained and multicolored protein markers can be produced that will serve as precise standards during SDS-PAGE. Likewise, purified proteins can be chemically modified using specific fluorescent reagents in order to generate a fluorescently labeled protein ladder, which may be useful when the fluorescence of a protein is required to be followed.…”

Section: Introductionmentioning

confidence: 99%

“…Prestained markers are also known to provide a convenient visual pattern of polypeptide bands with different apparent sizes on electrotransferred membranes in western blot experiments 2 . To prepare prestained standards, purified proteins can be covalently modified by various dyes, generating protein derivatives carrying different colors 3–7 . In this manner, a series of prestained and multicolored protein markers can be produced that will serve as precise standards during SDS‐PAGE.…”

Section: Introductionmentioning

confidence: 99%

Proposal of a laboratory course dedicated to the generation of protein molecular weight standards for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

Bubis

2020

Biochem Molecular Bio Educ

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Protein molecular weight standards are routinely employed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the apparent sizes of unknown proteins within a sample. For training students, a laboratory course based on the production of marker proteins is proposed. Following fractionation by column chromatography, a series of purified proteins and mixtures of proteins were combined to generate various sets of unstained, prestained and fluorescently labeled molecular weight ladders for SDS-PAGE. The produced material can be used in successive laboratory practices as an alternative to commercial protein markers.

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