Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethylmaleic anhydride prior to processing. Immobilon(TM) AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected ε-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzenesulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 μl) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.

Original languageEnglish (US)
Pages (from-to)45-53
Number of pages9
JournalJournal of Immunological Methods
Volume203
Issue number1
DOIs
StatePublished - Apr 11 1997

Fingerprint

Trinitrobenzenesulfonic Acid
Antibodies
Proteins
Membranes
Sample Size
Limit of Detection
Blood Proteins
Esters
Acetates
Monoclonal Antibodies

Keywords

  • 2,3-Dimethylmaleic anhydride
  • Covalent modification
  • Dot-blot assay
  • Immobilon AV

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

@article{05c4461c38d947bfb92bb990728ed284,
title = "Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups",
abstract = "The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethylmaleic anhydride prior to processing. Immobilon(TM) AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected ε-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzenesulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 μl) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.",
keywords = "2,3-Dimethylmaleic anhydride, Covalent modification, Dot-blot assay, Immobilon AV",
author = "T{\"u}lin Mor{\cc}{\"o}l and Anuradha Subramanian and Velander, {William H.}",
year = "1997",
month = "4",
day = "11",
doi = "10.1016/S0022-1759(97)00013-6",
language = "English (US)",
volume = "203",
pages = "45--53",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups

AU - Morçöl, Tülin

AU - Subramanian, Anuradha

AU - Velander, William H.

PY - 1997/4/11

Y1 - 1997/4/11

N2 - The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethylmaleic anhydride prior to processing. Immobilon(TM) AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected ε-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzenesulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 μl) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.

AB - The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethylmaleic anhydride prior to processing. Immobilon(TM) AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected ε-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzenesulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 μl) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.

KW - 2,3-Dimethylmaleic anhydride

KW - Covalent modification

KW - Dot-blot assay

KW - Immobilon AV

UR - http://www.scopus.com/inward/record.url?scp=0343554644&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0343554644&partnerID=8YFLogxK

U2 - 10.1016/S0022-1759(97)00013-6

DO - 10.1016/S0022-1759(97)00013-6

M3 - Article

C2 - 9134029

AN - SCOPUS:0343554644

VL - 203

SP - 45

EP - 53

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1

ER -