Characterization of a rabbit polyclonal antibody against threonine-AMPylation

Yi Heng Hao; Trinette Chuang; Haydn L. Ball; Phi Luong; Yan Li; Ruben D. Flores-Saaib; Kim Orth

doi:10.1016/j.jbiotec.2010.12.013

Characterization of a rabbit polyclonal antibody against threonine-AMPylation

Yi Heng Hao, Trinette Chuang, Haydn L. Ball, Phi Luong, Yan Li, Ruben D. Flores-Saaib, Kim Orth

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

Abstract

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.

Original language	English (US)
Pages (from-to)	251-254
Number of pages	4
Journal	Journal of Biotechnology
Volume	151
Issue number	3
DOIs	https://doi.org/10.1016/j.jbiotec.2010.12.013
State	Published - Feb 10 2011

Keywords

AMP
AMPylation
Antibody
Immunoprecipitation
Western blot

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology

Access to Document

10.1016/j.jbiotec.2010.12.013

Cite this

@article{b01c9441a3ab4e80be343ccb61802fbb,

title = "Characterization of a rabbit polyclonal antibody against threonine-AMPylation",

abstract = "An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.",

keywords = "AMP, AMPylation, Antibody, Immunoprecipitation, Western blot",

author = "Hao, {Yi Heng} and Trinette Chuang and Ball, {Haydn L.} and Phi Luong and Yan Li and Flores-Saaib, {Ruben D.} and Kim Orth",

note = "Funding Information: We thank members of the Orth lab for insightful discussions and critical reading. K.O., Y.-H.H., and P.L. are supported by grants from NIH-Allergy and Infectious Disease ( R01-AI056404 and R01-AI087808 ) and the Welch Foundation ( I-1561 ). K.O. is a Burroughs Wellcome Investigator in Pathogenesis of Infectious Disease and a W.W. Caruth, Jr. Biomedical Scholar.",

year = "2011",

month = feb,

day = "10",

doi = "10.1016/j.jbiotec.2010.12.013",

language = "English (US)",

volume = "151",

pages = "251--254",

journal = "Journal of Biotechnology",

issn = "0168-1656",

publisher = "Elsevier",

number = "3",

}

TY - JOUR

T1 - Characterization of a rabbit polyclonal antibody against threonine-AMPylation

AU - Hao, Yi Heng

AU - Chuang, Trinette

AU - Ball, Haydn L.

AU - Luong, Phi

AU - Li, Yan

AU - Flores-Saaib, Ruben D.

AU - Orth, Kim

N1 - Funding Information: We thank members of the Orth lab for insightful discussions and critical reading. K.O., Y.-H.H., and P.L. are supported by grants from NIH-Allergy and Infectious Disease ( R01-AI056404 and R01-AI087808 ) and the Welch Foundation ( I-1561 ). K.O. is a Burroughs Wellcome Investigator in Pathogenesis of Infectious Disease and a W.W. Caruth, Jr. Biomedical Scholar.

PY - 2011/2/10

Y1 - 2011/2/10

N2 - An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.

AB - An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.

KW - AMP

KW - AMPylation

KW - Antibody

KW - Immunoprecipitation

KW - Western blot

UR - http://www.scopus.com/inward/record.url?scp=79151471802&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79151471802&partnerID=8YFLogxK

U2 - 10.1016/j.jbiotec.2010.12.013

DO - 10.1016/j.jbiotec.2010.12.013

M3 - Article

C2 - 21185336

AN - SCOPUS:79151471802

SN - 0168-1656

VL - 151

SP - 251

EP - 254

JO - Journal of Biotechnology

JF - Journal of Biotechnology

IS - 3

ER -

Characterization of a rabbit polyclonal antibody against threonine-AMPylation

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this