The SCO2 gene encodes for a protein essential for the assembly and function of Mammalian cytochrome c oxidase (COX)(Complex IV) of the mitochondrial respiratory chain. SCO2 acts as a metallochaperone involved in the biogenesis of cytochrome c oxidase subunit II, an essential subunit of Complex IV which transfers the electrons from cytochrome c to the bimetallic center of the catalytic subunit 1 via its binuclear copper A center. The biogenesis involves the transport of copper to the Cu(A) site on the cytochrome c oxidase subunit II leading to the proper synthesis and maturation of the subunit. In addition, SCO2 acts as a thiol-disulfide oxidoreductase to regulate the redox state of the cysteines in SCO1 during maturation of the cytochrome c oxidase subunit II. The maturation and synthesis of cytochrome c oxidase subunit II is required for the function of Mammalian cytochrome c oxidase (COX)(Complex IV).Complex IV, a multimeric protein complex that requires several assembly factors, catalyzes the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane
Background References
1. Georgios C Kaiafas,Dionysia Papagiannopoulou,Αndroulla N. Miliotou, et al. In vivo biodistribution study of TAT-L-Sco2 fusion protein, developed as protein therapeutic for mitochondrial disorders attributed to SCO2 mutations. Molecular Genetics and Metabolism Reports. 2020;25 (0):100683-100683. doi:10.1016/j.ymgmr.2020.100683
2. Parthena Foltopoulou,Asterios S. Tsiftsoglou,Ioannis Bonovolias, et al. Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient\'s primary cells. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2010;1802 (6):497-508. doi:10.1016/j.bbadis.2010.02.009
Western blot analysis of SCO2 on different lysates with Rabbit anti-SCO2 antibody (HA723166) at 1/2,000 dilution.
Lane 1: MCF7 cell lysate Lane 2: HL-60 cell lysate Lane 3: SK-OV-3 cell lysate Lane 4: HepG2 cell lysate Lane 5: U-937 cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 30 kDa Observed band size: 30 kDa
Exposure time: 2 minutes; 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 (HA723166) 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.
Immunocytochemistry analysis of MCF7 cells labeling SCO2 with Rabbit anti-SCO2 antibody (HA723166) 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-SCO2 antibody (HA723166) 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 kidney tissue with Rabbit anti-SCO2 antibody (HA723166) 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 (HA723166) 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.
Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-SCO2 antibody (HA723166) 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 (HA723166) 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 kidney tissue with Rabbit anti-SCO2 antibody (HA723166) 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 (HA723166) 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.
SCO2 was immunoprecipitated from 0.2 mg HepG2 cell lysate with HA723166 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723166 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: HepG2 cell lysate (input) Lane 2: HA723166 IP in HepG2 cell lysate Lane 3: Rabbit IgG instead of HA723166 in HepG2 cell lysate