Generation of polyclonal antiserum for the detection of methylarginine proteins

Peng Duan, Ye Xu, Barbara Birkaya, Jason Myers, Michel Pelletier, Laurie K. Read, Corrado Guarnaccia, Sandor Pongor, Robert B. Denman, John M. Aletta

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

This report describes an approach for the study of the biology of methylarginine proteins based on the generation of immunological reagents capable of recognizing the methylarginine status of cellular proteins. Two forms of an immunizing peptide were prepared based upon an amino acid sequence motif found most prevalently among verified dimethylarginine-containing proteins. One form of the peptide was constructed with 7 arginine residues alternating with 8 glycine residues. None of the arginines used in the synthesis were methylated. The alternative form of the peptide was synthesized with the identical repeating GRG sequence, but with asymmetrical dimethylarginine at each arginine residue. A methylarginine-specific antiserum was generated using the latter peptide. ELISA and western blotting of glycine arginine-rich peptides, each synthesized with or without asymmetric dimethylarginine, demonstrate the methyl specificity of the antiserum. The methylarginine-specific antibody co-localizes with the highly methylated native nucleolin protein conspicuously concentrated in the nucleolus. The methylarginine-specific antiserum recognizes a GRG peptide and bacterially expressed RBP16 only after incubation of the peptide or RBP16 with recombinant protein arginine methyltransferase 1, or cell extracts, respectively. Proteins isolated from cells in different developmental states exhibit different patterns of reactivity observed by western blots. Finally, the methylarginine-specific reagent interacts specifically with the methylarginine of cellular hnRNPA1 and human fragile X mental retardation protein expressed in cultured PC12 cells. An immunological reagent capable of detecting the methylarginine status of cellular methylproteins will facilitate the cellular and molecular analysis of protein arginine methylation in a wide variety of research and biomedical applications.

Original languageEnglish (US)
Pages (from-to)132-142
Number of pages11
JournalJournal of Immunological Methods
Volume320
Issue number1-2
DOIs
StatePublished - Mar 30 2007
Externally publishedYes

Fingerprint

Immune Sera
Arginine
Peptides
Proteins
Glycine
Protein-Arginine N-Methyltransferases
Fragile X Mental Retardation Protein
Western Blotting
Amino Acid Motifs
PC12 Cells
Cell Extracts
Recombinant Proteins
Methylation
Biomedical Research
Amino Acid Sequence
Cultured Cells
Enzyme-Linked Immunosorbent Assay
Antibodies

Keywords

  • Asymmetrical dimethylarginine
  • FMRP
  • Nucleolin
  • Peptide synthesis

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Duan, P., Xu, Y., Birkaya, B., Myers, J., Pelletier, M., Read, L. K., ... Aletta, J. M. (2007). Generation of polyclonal antiserum for the detection of methylarginine proteins. Journal of Immunological Methods, 320(1-2), 132-142. https://doi.org/10.1016/j.jim.2007.01.006

Generation of polyclonal antiserum for the detection of methylarginine proteins. / Duan, Peng; Xu, Ye; Birkaya, Barbara; Myers, Jason; Pelletier, Michel; Read, Laurie K.; Guarnaccia, Corrado; Pongor, Sandor; Denman, Robert B.; Aletta, John M.

In: Journal of Immunological Methods, Vol. 320, No. 1-2, 30.03.2007, p. 132-142.

Research output: Contribution to journalArticle

Duan, P, Xu, Y, Birkaya, B, Myers, J, Pelletier, M, Read, LK, Guarnaccia, C, Pongor, S, Denman, RB & Aletta, JM 2007, 'Generation of polyclonal antiserum for the detection of methylarginine proteins', Journal of Immunological Methods, vol. 320, no. 1-2, pp. 132-142. https://doi.org/10.1016/j.jim.2007.01.006
Duan, Peng ; Xu, Ye ; Birkaya, Barbara ; Myers, Jason ; Pelletier, Michel ; Read, Laurie K. ; Guarnaccia, Corrado ; Pongor, Sandor ; Denman, Robert B. ; Aletta, John M. / Generation of polyclonal antiserum for the detection of methylarginine proteins. In: Journal of Immunological Methods. 2007 ; Vol. 320, No. 1-2. pp. 132-142.
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