The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 83 kDa (predicted molecular weight: 83 kDa).
Use a concentration of 1 µg/ml.
Lipid metabolism; fatty acid beta-oxidation.
Defects in HADHA are a cause of trifunctional protein deficiency (TFP deficiency) [MIM:609015]. The clinical manifestations are very variable and include hypoglycemia, cardiomyopathy and sudden death. Phenotypes with mainly hepatic and neuromyopathic involvement can also be distinguished. Biochemically, TFP deficiency is defined by the loss of all enzyme activities of the TFP complex. Defects in HADHA are the cause of long-chain 3-hydroxyl-CoA dehydrogenase deficiency (LCHAD deficiency) [MIM:609016]. The clinical features are very similar to TFP deficiency. Biochemically, LCHAD deficiency is characterized by reduced long-chain 3-hydroxyl-CoA dehydrogenase activity, while the other enzyme activities of the TFP complex are normal or only slightly reduced. Defects in HADHA are a cause of maternal acute fatty liver of pregnancy (AFLP) [MIM:609016]. AFLP is a severe maternal illness occurring during pregnancies with affected fetuses. This disease is associated with LCHAD deficiency and characterized by sudden unexplained infant death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome).
In the N-terminal section; belongs to the enoyl-CoA hydratase/isomerase family. In the central section; belongs to the 3-hydroxyacyl-CoA dehydrogenase family.
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: HADHA knockout HAP1 cell lysate (20 µg) Lane 3: HEK293 cell lysate (20 µg) Lane 4: HepG2 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab93207 observed at 85 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab93207 was shown to specifically react with HADHA when HADHA knockout samples were used. Wild-type and HADHA knockout samples were subjected to SDS-PAGE. ab93207 and ab8245 (loading control to GAPDH) were diluted 1 µg/mL and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
ICC/IF image of ab93207 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab93207, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
Western blot - HADHA antibody (ab93207)
All lanes : Anti-HADHA antibody (ab93207) at 1 µg/ml
Lane 1 : Human liver tissue lysate - total protein (ab29889) Lane 2 : Human testis tissue lysate - total protein (ab30257) Lane 3 : Human kidney tissue lysate - total protein (ab30203) Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 6 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 7 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 83 kDa Observed band size : 83 kDa Additional bands at : 50 kDa. We are unsure as to the identity of these extra bands.