Mouse anti-HA Tag Monoclonal Antibody (Clone KT25)

Immunofluorescence analysis of HEK-293 cells either transfected with a construct expressing HA tagged ARF3 (Cat. P61801QETN) at the N-terminus (A) or untransfected (B). The cells were fixed, permeabilized, blocked and incubated with 10 ug/ml of KT25 as the primary antibody. FITC-labeled goat anti-mouse IgG antibody (green) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue). Result: KT25 can detect HA tag by Immunofluorescence.

Immunofluorescence analysis of HEK-293 cells either transfected with a construct expressing HA tagged CDA (Cat. P60351QETN) at the C-terminus (A) or untransfected (B). The cells were fixed, permeabilized, blocked and incubated with 10 ug/ml of KT25 as the primary antibody. FITC-labeled goat anti-mouse IgG antibody (green) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue). Result: KT25 can detect HA tag by Immunofluorescence.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of KT25 with 200 ug of cell lysate extracted from HEK-293 transfected with HA 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. HA tag antibody (KT25) 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 HA tagged ARF3 (Cat. P61801QETN) at N-terminus) Lane 2: HA tagged protein immunoprecipitated by KT25 from the same lysate as Lane 1 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: KT25 can immunoprecipitate HA tag.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of KT25 with 200 ug of cell lysate extracted from HEK-293 transfected with HA 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. HA tag antibody (KT25) 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 HA tagged CDA (Cat. P60351QETN) at C-terminus) Lane 2: HA tagged protein immunoprecipitated by KT25 from the same lysate as Lane 1 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: KT25 can immunoprecipitate HA tag.

Immunoprecipitation: Immunoprecipitation was performed by incubation of 2.5 ug of KT25 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. HA tag antibody (KT25) 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 KT25 Lane 3: The same as Lane 2 but KT82 was used as IgG isotype control antibody Result: Unspecific immunoprecipitation by KT25 was not observed.

Western Blot: Various protein samples were run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. KT25 at 0.5 ug/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. HA tagged protein bands were visualized using ECL Western Blotting Substrate. Lane 1: 10 ug of HEK-293 lysate (untransfected) Lane 2: 10 ug of HEK-293 lysate (transfected with a construct expressing HA tagged CDA (Cat. P60351QETN) at C-terminus) Lane 3: 10 ug of HEK-293 lysate (transfected with a construct expressing HA tagged ARF3 (Cat. P61801QETN) at N-terminus) Result: KT25 can detect HA tag by Western blotting.