Characterization of HIV Tat modifications using novel methyl-lysine-specific antibodies

Methods. 2011 Jan;53(1):91-6. doi: 10.1016/j.ymeth.2010.07.001. Epub 2010 Jul 6.

Abstract

Modification-specific antibodies are important tools to examine the dynamics and functions of posttranslational protein modifications in cells. Here, we describe in detail the generation of polyclonal antibodies specific for mono-, di-, and trimethylated lysine 51 within the HIV transactivator Tat. Lysine 51 is a highly conserved residue located in the RNA-binding region of Tat and the target of lysine methyltransferases KMT1E (SETDB1) and KMT7 (Set7/9). Using affinity-purified methyl-specific antibodies of Tat, we find that cellular Tat is predominantly monomethylated at lysine 51, a modification enhanced by coexpression of KMT7.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies*
  • Blotting, Western
  • HEK293 Cells
  • HIV-1 / chemistry*
  • Humans
  • Immunoblotting
  • Lysine / analogs & derivatives*
  • Lysine / chemistry
  • Lysine / immunology
  • Methylation
  • Protein Processing, Post-Translational
  • Rabbits
  • Transfection
  • tat Gene Products, Human Immunodeficiency Virus / chemistry*
  • tat Gene Products, Human Immunodeficiency Virus / immunology

Substances

  • Antibodies
  • tat Gene Products, Human Immunodeficiency Virus
  • epsilon-N-methyllysine
  • Lysine