Mouse anti-DYKDDDDK (FLAG) Tag Monoclonal Antibody (Clone K11016_5D8)

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of K11016_5D8 with 200 ug of cell lysate extracted from HEK-293 transfected with DYKDDDDK tagged ARF3 at N-terminus. After absorption with Protein G beads, the mixture was run on 6-18% SDS-PAGE and blotted onto nitrocellulose membrane. DYKDDDDK tag antibody (K11016_5D8) at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG (Fc specific) was used as the secondary antibody. The isotype control antibody was KT82. Lane 1: HEK-293 lysate (transfected with DYKDDDDK tagged ARF3 (Cat. P61801QBTN) at N-terminus) Lane 2: DYKDDDDK tagged protein immunoprecipitated by K11016_5D8 from the same lysate as Lane 1 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: K11016_5D8 can immunoprecipitate DYKDDDDK tag.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of K11016_5D8 with 200 ug of cell lysate extracted from HEK-293 transfected with DYKDDDDK tagged CDA at C-terminus. After absorption with Protein G beads, the mixture was run on 6-18% SDS-PAGE and blotted onto nitrocellulose membrane. DYKDDDDK tag antibody (K11016_5D8) at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG (Fc specific) was used as the secondary antibody. The isotype control antibody was KT82. Lane 1: HEK-293 lysate (transfected with DYKDDDDK tagged CDA (Cat. P60351QBTN) at C-terminus) Lane 2: DYKDDDDK tagged protein immunoprecipitated by K11016_5D8 from the same lysate as Lane 1 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: K11016_5D8 can immunoprecipitate DYKDDDDK tag.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of K11016_5D8 with untransfected HEK-293 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. DYKDDDDK tag antibody (K11016_5D8) at 1 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG (Fc specific) was used as the secondary antibody. The isotype control antibody was KT82. Lane 1: Untransfected HEK-293 lysate Lane 2: Sample immunoprecipitated from untransfected HEK-293 lysate by K11016_5D8 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: Unspecific immunoprecipitation by K11016_5D8 was not observed.

Indirect ELISA96-well microtitre plates were coated with 1 ug/mL of flag-tagged recombinant PD-L1 and BCMA at 4 C overnight. Plates were blocked with 2 % BSA in PBS-T (0.1 % Tween) at room temperature for 1 hour. Serial diluted of K11016_5D8 was used as the primary antibody and added into the plates for 1 hour incubation. Plates were washed with PBS-T for three times and then incubated with HRP conjugated goat-anti-mouse IgG (Fc specific) for 1 hour. After washed with PBS-T for three times, the plates were incubated with ABTS for 15 minute. The absorbance (optical density) was read with a plate reader.

Western Blot: Various protein samples were run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. K11016_5D8 at 0.5 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. DYKDDDDK tagged protein bands were visualized using ECL Western Blotting Substrate. Lane 1: 15 ug of HEK-293 lysate (untransfected) Lane 2: 15 ug of HEK-293 lysate (transfected with a construct expressing DYKDDDDK tagged CDA (Cat. P60351QBTN) at C-terminus) Lane 3: 15 ug of HEK-293 lysate (transfected with a construct expressing DYKDDDDK tagged ARF3 (Cat. P61801QBTN) at N-terminus) Result: K11016_5D8 can detect DYKDDDDK tag by Western blotting.