Mouse anti-Crotonyl Lysine Monoclonal Antibody (Clone K16287_3A9)

Dot blotting: Various peptides were loaded on nitrocellulose membrane. The membrane was blocked with 5% non-fat milk. K16287_3A9 at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Lane 1: Lys (crotonylated) peptides library Lane 2: Lys (lactated) peptides library Lane 3: Lys peptides library Result: K16287_3A9 can detect crotonylated lysine peptides by dot blotting.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of K16287_3A9 with crotonic acid treated HeLa lysate containing 200 ug of total protein. After absorption with Protein G beads, the mixture was run on 6-18% SDS-PAGE and blotted onto nitrocellulose membrane. OC-180 at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG (light chain specific for membrane above 43 kDa and heavy chain specific for membrane below 43 kDa) was used as the secondary antibody. The isotype control antibody was KT82. Lane 1: HeLa lysate treated with crotonic acid (10 mM, 16 h) Lane 2: Crotonyl Lysine immunoprecipitated by K16287_3A9 from the same lysate as Lane 1 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: K16287_3A9 can immunoprecipitate crotonylated proteins.

Western Blot: HeLa cells treated with or without crotonic acid. Lysates were run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. K16287_3A9 at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Crotonylated protein bands were visualized using ECL Western Blotting Substrate. Lane 1: 15 ug of HeLa lysate Lane 2: 15 ug of crotonic acid (10mM, 16h) treated HeLa lysate Result: K16287_3A9 can detect crotonylated proteins by Western blotting.