Very long-chain specific acyl-CoA dehydrogenase is one of the acyl-CoA dehydrogenases that catalyze the first step of mitochondrial fatty acid beta-oxidation, an aerobic process breaking down fatty acids into acetyl-CoA and allowing the production of energy from fats.
The first step of fatty acid beta-oxidation consists in the removal of one hydrogen from C-2 and C-3 of the straight-chain fatty acyl-CoA thioester, resulting in the formation of trans-2-enoyl-CoA. Among the different mitochondrial acyl-CoA dehydrogenases, very long-chain specific acyl-CoA dehydrogenase acts specifically on acyl-CoAs with saturated 12 to 24 carbons long primary chains. Predominantly expressed in heart and skeletal muscle (at protein level). Also detected in kidney and liver (at protein level).
Background References
1. Aoyama T., Souri M., Ueno I., Kamijo T., Yamaguchi S., Rhead W.J., Tanaka K., Hashimoto T. Cloning of human very-long-chain acyl-coenzyme A dehydrogenase and molecular characterization of its deficiency in two patients. Am. J. Hum. Genet. 57:273-283 (1995).
2. Souri M., Aoyama T., Yamaguchi S., Hashimoto T. Relationship between structure and substrate-chain-length specificity of mitochondrial very-long-chain acyl-coenzyme A dehydrogenase. Eur. J. Biochem. 257:592-598 (1998)
Western blot analysis of ACADVL / VLCAD on different lysates with Rabbit anti-ACADVL / VLCAD antibody (HA722416) at 1/1,000 dilution.
Lane 1: HEK-293 cell lysate Lane 2: A431 cell lysate Lane 3: HepG2 cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 70 kDa Observed band size: 70 kDa
Exposure time: 59 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 (HA722416) 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.
Immunocytochemistry analysis of A431 cells labeling ACADVL / VLCAD with Rabbit anti-ACADVL / VLCAD antibody (HA722416) 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-ACADVL / VLCAD antibody (HA722416) 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 (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.
Immunohistochemical analysis of paraffin-embedded human small intestine tissue with Rabbit anti-ACADVL / VLCAD antibody (HA722416) 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 (HA722416) 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.
ACADVL / VLCAD was immunoprecipitated from 0.2 mg A431 cell lysate with HA722416 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722416 at 1/1,000 dilution. Anti-Rabbit IgG for IP, AlpSdAbs® VHH(HRP) (025-100-005) at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: A431 cell lysate (input) Lane 2: HA722416 IP in A431 cell lysate Lane 3: Rabbit IgG instead of HA722416 in A431 cell lysate