alpha Internexin Recombinant Rabbit Monoclonal Antibody [JU25-03]

☑ Relative expression (RE)

Western blot analysis of alpha Internexin on different lysates with Rabbit anti-alpha Internexin antibody (ET1706-23) at 1/2,000 dilution.

Lane 1: SH-SY5Y cell lysate
Lane 2: Human brain tissue lysate
Lane 3: Mouse brain tissue lysate
Lane 4: Rat brain tissue lysate
Lane 5: Mouse pancreas tissue lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 55 kDa
Observed band size: 60 kDa

Exposure time: 6 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 (ET1706-23) at 1/2,000 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.

☑ Knockdown (KD)

Western blot analysis of alpha Internexin on different lysates with Rabbit anti-alpha Internexin antibody (ET1706-23) at 1/5,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-alpha Internexin KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 55 kDa
Observed band size: 60 kDa

Exposure time: 180 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 (ET1706-23) at 1/5,000 dilution was used in 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.

Immunocytochemistry analysis of SH-SY5Y cells labeling alpha Internexin with Rabbit anti-alpha Internexin antibody (ET1706-23) 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-alpha Internexin antibody (ET1706-23) 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 human brain tissue with Rabbit anti-alpha Internexin antibody (ET1706-23) at 1/200 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 (ET1706-23) at 1/200 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.

Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-alpha Internexin antibody (ET1706-23) at 1/200 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 (ET1706-23) at 1/200 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.

Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-alpha Internexin antibody (ET1706-23) at 1/200 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 (ET1706-23) at 1/200 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.

Immunofluorescence analysis of paraffin-embedded rat brain tissue labeling alpha Internexin with Rabbit anti-alpha Internexin antibody (ET1706-23) 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1706-23, green) at 1/50 dilution overnight at 4 ℃, washed with PBS.

Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Flow cytometric analysis of SH-SY5Y cells labeling alpha Internexin.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1706-23, 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).

alpha Internexin was immunoprecipitated from 0.2 mg SH-SY5Y cell lysate with ET1706-23 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1706-23 at 1/2,000 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: SH-SY5Y cell lysate (input)
Lane 2: ET1706-23 IP in SH-SY5Y cell lysate
Lane 3: Rabbit IgG instead of ET1706-23 in SH-SY5Y cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 12 seconds; ECL: K1801