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☑ Relative expression (RE)
Western blot analysis of Lin28A on different lysates with Rabbit anti-Lin28A antibody (HA722899) at 1/1,000 dilution.
Lane 1: NCCIT cell lysate
Lane 2: JAR cell lysate
Lane 3: HeLa cell lysate (negative)
Lysates/proteins at 20 µg/Lane.
Predicted band size: 23 kDa
Observed band size: 29 kDa
Exposure time: 4 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 (HA722899) at 1/1,000 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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☑ Relative expression (RE)
Immunocytochemistry analysis of NCCIT (positive) and HeLa (negative) labeling Lin28A with Rabbit anti-Lin28A antibody (HA722899) at 1/500 dilution.
Cells were fixed in 4% paraformaldehyde for 15 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-Lin28A antibody (HA722899) at 1/500 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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Flow cytometric analysis of NCCIT cells labeling Lin28A.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722899, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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Lin28A was immunoprecipitated from 0.2 mg JAR cell lysate with HA722899 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722899 at 1/1,000 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: JAR cell lysate (input)
Lane 2: HA722899 IP in JAR cell lysate
Lane 3: Rabbit IgG instead of HA722899 in JAR cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 6 seconds; ECL: K1801
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