mtTFA Recombinant Rabbit Monoclonal Antibody [JM32-44]

Western blot analysis of mtTFA on different lysates with Rabbit anti-mtTFA antibody (ET1705-64) at 1/1,000 dilution.

Lane 1: K-562 cell lysate (20 µg/Lane)
Lane 2: HL-60 cell lysate (20 µg/Lane)
Lane 3: HeLa cell lysate (20 µg/Lane)
Lane 4: HepG2 cell lysate (20 µg/Lane)
Lane 5: MCF7 cell lysate (20 µg/Lane)
Lane 6: Human brain tissue lysate (40 µg/Lane)

Predicted band size: 29 kDa
Observed band size: 24 kDa

Exposure time: 1 minute; 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 (ET1705-64) 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.

Immunofluorescence analysis of paraffin-embedded human kidney tissue labeling mtTFA with Rabbit anti-mtTFA antibody (ET1705-64) 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1705-64, green) at 1/200 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).

Immunofluorescence analysis of paraffin-embedded human colon tissue labeling mtTFA with Rabbit anti-mtTFA antibody (ET1705-64) 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1705-64, green) at 1/200 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).

Immunocytochemistry analysis of HeLa cells labeling mtTFA with Rabbit anti-mtTFA antibody (ET1705-64) at 1/100 dilution.

Cells 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-mtTFA antibody (ET1705-64) 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. Counterstained with Mitotracker. Nuclear DNA was labelled in blue with DAPI.

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-mtTFA antibody (ET1705-64) at 1/1,000 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 (ET1705-64) at 1/1,000 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.

mtTFA was immunoprecipitated from 0.2 mg K-562 cell lysate with ET1705-64 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1705-64 at 1/1,000 dilution. Mouse anti Rabbit IgG heavy chain (Fc) secondary antibody (M1003-7) at 1/100,000 dilution was used for 1 hour at room temperature.

Lane 1: K-562 cell lysate (input)
Lane 2: ET1705-64 IP in K-562 cell lysate
Lane 3: Rabbit K-562 instead of ET1705-64 in 293T cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute; ECL: K1801