V5 tag Recombinant Rabbit Monoclonal Antibody [SY30-01]

Western blot analysis of V5 tag on different lysates with Rabbit anti-V5 tag antibody (ET1605-41) at 1/5,000 dilution.

Lane 1: 293T cell lysate
Lane 2: 293T transfected with V5-tagged Histone H3.1 (N-terminal) cell lysate
Lane 3: 293T transfected with V5-tagged Nanos homolog 3 (C-terminal) cell lysate

Lysates/proteins at 10 µg/Lane.

Exposure time: 2 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1605-41) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of HeLa cells labeling V5 tag with Rabbit anti-V5 tag antibody (ET1605-41) at 1/2,000 dilution.

HeLa cells, transfected with V5-tagged empty control, Histone H3 (N-terminal) or Nanos homolog 3 (C-terminal) expression vector, respectively, were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-V5 tag antibody (ET1605-41) at 1/2,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

V5 tag (M1008-2, red) was stained at 1/1,000 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

V5 tag was immunoprecipitated from 0.2 mg 293T transfected with V5-tagged Histone H3.1 (N-terminal) cell lysate with ET1605-41 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using ET1605-41 at 1/10,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: 293T transfected with V5-tagged Histone H3.1 (N-terminal) cell lysate (input)
Lane 2: ET1605-41 IP in 293T transfected with V5-tagged Histone H3.1 (N-terminal) cell lysate
Lane 3: Rabbit IgG instead of ET1605-41 in 293T transfected with V5-tagged Histone H3.1 (N-terminal) cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 seconds; ECL: K1801