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 75 kDa (predicted molecular weight: 70 kDa).
Use a concentration of 5 µg/ml.
RelevanceThrombin is an active enzyme in the earliest steps of the blood clot formation, generated from its circulating inactive precursor prothrombin. Thrombin is a glycoprotein formed by two peptides chains of 36 and 259 amino acids linked by disulfure bonds. Three important sites have been identified on the surface of the enzyme: The catalytic site that confers to the molecule its serine protease activity, the exosite one responsible for the binding of the substrate (fibrinogen or thrombin receptor) and the exosite two responsible for the binding of antithrombin III and inactivation of thrombin. The molecular mass of thrombin is 37 kDa.
All lanes : Anti-Thrombin antibody (ab92621) at 1 µg/ml
Lane 1 : Human liver tissue lysate - total protein (ab29889) Lane 2 : Liver (Mouse) Tissue Lysate Lane 3 : Liver (Rat) Tissue Lysate Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 5 : Human Plasma Total Protein 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
Exposure time : 10 minutesThrombin is expressed by liver and secreted in plasma. The target sequence contains three potential glycosylation sites and a 24 residue signal sequence (SwissProt Data). This may explain the banding pattern observed.
ICC/IF image of ab92621 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 (ab92621, 5µg/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) HeLa, Hek293 and MCF7 cells at 5µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
References for Anti-Thrombin antibody (ab92621)
This product has been referenced in:
Pan XY et al. Hirudin promotes angiogenesis by modulating the cross-talk between p38 MAPK and ERK in rat ischemic skin flap tissue. Tissue Cell47:301-10 (2015).
Read more (PubMed: 25958163) »
Chen J et al. Antithrombin nanoparticles improve kidney reperfusion and protect kidney function after ischemia-reperfusion injury. Am J Physiol Renal Physiol308:F765-73 (2015).
Read more (PubMed: 25651565) »