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

Methods (San Diego, Calif.) (2010-07-10)
Sara Pagans, Naoki Sakane, Martina Schnölzer, Melanie Ott
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-monomethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-dimethyl-Histone H3 (Lys9) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-trimethyl-Histone H3 (Lys9) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-α-Tubulin antibody, Mouse monoclonal, clone B-5-1-2, purified from hybridoma cell culture
Sigma-Aldrich
Nε-Methyl-L-lysine hydrochloride, ≥98.0% (TLC)
Sigma-Aldrich
Anti-dimethyl-Histone H3 (Lys4) Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-Histone H3 Antibody, CT, pan, serum, Upstate®