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Western blot analysis of Strep-Tag II on different lysates with Rabbit anti-Strep-Tag II antibody (HA722229) at 1/10,000 dilution.
Lane 1: 293T transfected with Strep-Tag II-tagged Histone H3.1 (C-terminal) cell lysate
Lane 2: 293T transfected with Strep-Tag II-tagged ACAT2 (N-terminal) cell lysate
Lysates/proteins at 10 µg/Lane.
Exposure time: 2 seconds;
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 (HA722229) at 1/10,000 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.
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Immunocytochemistry analysis of HeLa cells labeling Strep-Tag II with Rabbit anti-Strep-Tag II antibody (HA722229) at 1/2,500 dilution.
HeLa cells, transfected with Strep-Tag II-tagged Histone H3.1 (C-terminal) or ACAT2 (N-terminal) expression vector, respectively, were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-Strep-Tag II antibody (HA722229) at 1/2,500 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.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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Strep-Tag II was immunoprecipitated from 0.2 mg 293T transfected with Strep-Tag II-tagged Histone H3.1 (C-terminal) cell lysate with HA722229 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722229 at 1/5,000 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 Strep-Tag II-tagged Histone H3.1 (C-terminal) cell lysate (input)
Lane 2: HA722229 IP in 293T transfected with Strep-Tag II-tagged Histone H3.1 (C-terminal) cell lysate
Lane 3: Rabbit IgG instead of HA722229 in 293T transfected with Strep-Tag II-tagged Histone H3.1 (C-terminal) cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 seconds; ECL: K1801
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Strep-Tag II was immunoprecipitated from 0.2 mg 293T transfected with Strep-Tag II-tagged ACAT2 (N-terminal) cell lysate with HA722229 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722229 at 1/5,000 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 Strep-Tag II-tagged ACAT2 (N-terminal) cell lysate (input)
Lane 2: HA722229 IP in 293T transfected with Strep-Tag II-tagged ACAT2 (N-terminal) cell lysate
Lane 3: Rabbit IgG instead of HA722229 in 293T transfected with Strep-Tag II-tagged ACAT2 (N-terminal) cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 seconds; ECL: K1801
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