LINE-1 ORF1p Recombinant Rabbit Monoclonal Antibody [PSH18-99]

☑ Relative expression (RE)

Western blot analysis of LINE-1 ORF1p on different lysates with Rabbit anti-LINE-1 ORF1p antibody (HA724035) at 1/1,000 dilution.

Lane 1: F9 cell lysate (20 µg/Lane)
Lane 2: NIH/3T3 cell lysate (negative) (20 µg/Lane)

Predicted band size: 41 kDa
Observed band size: 41 kDa

Exposure time: 40 seconds; ECL: K1802;

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 (HA724035) at 1/1,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Relative expression (RE)

Immunocytochemistry analysis of F9 (positive) and NIH/3T3 (negative) labeling LINE-1 ORF1p with Rabbit anti-LINE-1 ORF1p antibody (HA724035) at 1/100 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-LINE-1 ORF1p antibody (HA724035) at 1/100 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 (HA601187, 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.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue with Rabbit anti-LINE-1 ORF1p antibody (HA724035) at 1/50 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA724035) at 1/50 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

☑ Relative expression (RE)

Flow cytometric analysis of NIH/3T3 (left, negative) and F9 (right, positive) cells labeling LINE-1 ORF1p.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA724035, 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).

LINE-1 ORF1p was immunoprecipitated from 0.2 mg F9 cell lysate with HA724035 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724035 at 1/1,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: F9 cell lysate (input)
Lane 2: HA724035 IP in F9 cell lysate
Lane 3: Rabbit IgG instead of HA724035 in F9 cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 3 minutes; ECL: K1801