Mouse anti-Phospho Tyrosine Monoclonal Antibody (Clone K06352_10A10)

Dot blotting: Various peptides were loaded on nitrocellulose membrane. The membrane was blocked with 5% non-fat milk. K06352_10A10 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: pTyr peptides library Lane 2: pThr peptides library Lane 3: pSer peptides library Lane 4: Tyr peptides library Lane 5: Thr peptides library Lane 6: Ser peptides library Result: K06352_10A10 can detect phospho tyrosine peptides by dot blotting.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of K06352_10A10 with sodium vanadate 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-178 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 for membrane blow 43 kDa) was used as the secondary antibody. The isotype control antibody was KT82. Lane 1: HeLa lysate treated with sodium vanadate (1 mM, 45 min) Lane 2: Phospho Tyrosine immunoprecipitated by K06352_10A10 from the same lysate as Lane 1 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: K06352_10A10 can immunoprecipitate proteins with phospho tyrosine modification.

Western Blot: HeLa cells treated with or without 1 mM sodium vanadate for 45 min. Lysates were run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. K06352_10A10 at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Proteins with phospho tyrosine modifications were visualized using ECL Western Blotting Substrate. Lane 1: 15 ug of HeLa lysate Lane 2: 15 ug of sodium vanadate treated HeLa lysate Result: K06352_10A10 can detect proteins with phospho tyrosine modification by Western blotting.