Mouse anti-Lactyl Lysine Monoclonal Antibody (Clone K16286_5G3)

Dot blotting: Various peptides were loaded on nitrocellulose membrane. The membrane was blocked with 5% non-fat milk. K16286_5G3 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: K16286_5G3 can detect lactyl lysine peptides by dot blotting.

Dot blotting-block: Various peptides were loaded on nitrocellulose membrane. The membrane was blocked with 5% non-fat milk. K16286_5G3 was blocked with different types of antigens (molar ratio: 1:100) at room temperature for 1 hour. The membrane was then incubated with the blocked antibody for 1 hour. Peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Lane 1: Lys peptides library Lane 2: Lys (lactated) peptides library Lane 3: Lys (crotonylated) peptides library Membrane A: Antibody incubated with PBS Membrane B: Antibody incubated with lysine peptides library Membrane C: Antibody incubated with lactyl lysine peptides library Result: K16286_5G3 can be specifically blocked by lactyl lysine peptides.

Western Blot: HeLa cells treated with or without sodium lactate. Lysates were run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. K16286_5G3 at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Lactyl protein bands were visualized using ECL Western Blotting Substrate. Lane 1: 15 ug of HeLa lysate Lane 2: 15 ug of sodium lactate treated (50 mM, 24 h) HeLa lysate Result: K16286_5G3 can detect lactyl proteins by Western blotting