Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [6F7] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [6F7] - BSA and Azide free (AB255980)

ab213519 was incubated with 100ug HEK-293 PDL-1-DDDDK tagged overexpression lysate overnight at 4°C before being mixed with Protein G beads and precipitated on the ThermoFisher KingFisher Flex® using the indirect method. Protein was eluted from the beads with LDS-based loading buffer before being run in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween-20®) before incubation with Anti-DDDDK tag antibody [M2] (HRP) (ab49764). Antibody binding was detected using ECL development solution (ab133406).

Lane 1 : HeLa whole cell lysate (10 μg) Input - 10%

Lane 2 : Beads only (20μL)

Lane 3 : ab213519 (1μg/mL) IP in HEK-293 PDL-1-DDDDK tagged overexpression lysate (20μL)

Lane 4 : ab213519 (5μg/mL) IP in HEK-293 PDL-1-DDDDK tagged overexpression lysate (20μL)

All lanes:

Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [6F7] (<a href='/en-us/products/primary-antibodies/ddddk-tag-binds-to-flag-tag-sequence-antibody-6f7-ab213519'>ab213519</a>)

false

Exposure time: 30s

• ELISA

Lab

ELISA - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [6F7] - BSA and Azide free (AB255980)

96-well microtitre plates were coated overnight at 4°C with Recombinantn human RANKL protein (Active) (ab157289) in duplicate at concentrations of 1 and 0.1 μg/mL. Plates were blocked with 1% BSA in PBS-T (0.1% Tween^®) for 1 hour before incubation with an 8-step 4x serial dilution of ab213519 from 10 μg/mL for 1 hour at room temperature. Antibody binding was detected with Goat Anti-Rat IgG H&L (HRP) (ab97057) secondary antibody at a 1 in 10000 dilution for 1 hour at room temperature.

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [6F7] - BSA and Azide free (AB255980)

All lanes:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [6F7] (<a href='/en-us/products/primary-antibodies/ddddk-tag-binds-to-flag-tag-sequence-antibody-6f7-ab213519'>ab213519</a>) at 1 µg/mL

Lanes 1 and 3:

Western blot - Recombinant human RANKL protein (Active) (<a href='/en-us/products/proteins-peptides/recombinant-human-rankl-protein-active-ab157289'>ab157289</a>) at 0.5 µg with Milk in TBS-T

Lanes 2 and 4:

Western blot - Recombinant human RANKL protein (Active) (<a href='/en-us/products/proteins-peptides/recombinant-human-rankl-protein-active-ab157289'>ab157289</a>) at 0.1 µg with Milk in TBS-T

Lane 3:

Western blot - Recombinant human TRAIL protein (<a href='/en-us/products/proteins-peptides/recombinant-human-trail-protein-ab157345'>ab157345</a>) at 0.5 µg with Milk in TBS-T

Lane 4:

Western blot - Recombinant human TRAIL protein (<a href='/en-us/products/proteins-peptides/recombinant-human-trail-protein-ab157345'>ab157345</a>) at 0.1 µg with Milk in TBS-T

Secondary

All lanes:

Goat Anti-Rat IgG H&L (IRDye® 800CW ) (ab253031) at 1/20000 dilution

false