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.
FunctionThrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing.
Tissue specificityExpressed by the liver and secreted in plasma.
Involvement in diseaseFactor II deficiency Ischemic stroke Thrombophilia due to thrombin defect Pregnancy loss, recurrent, 2
Post-translational modificationsThe gamma-carboxyglutamyl residues, which bind calcium ions, result from the carboxylation of glutamyl residues by a microsomal enzyme, the vitamin K-dependent carboxylase. The modified residues are necessary for the calcium-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin. N-glycosylated. N-glycan heterogeneity at Asn-121: Hex3HexNAc3 (minor), Hex4HexNAc3 (minor) and Hex5HexNAc4 (major). At Asn-143: Hex4HexNAc3 (minor) and Hex5HexNAc4 (major).
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) »