The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/2000 - 1/10000. Predicted molecular weight: 187 kDa. Detects a strong band of approximately 9 kDa and two weaker bands at 115 and 25 - 36 kDa. Detection of the 9 kD band is improved by using PVDF membrane with 0.2um pores instead of 0.45um, and a Tricine gel buffer instead of Tris/glycine, according to customer feedback.
1/2000 - 1/10000.
Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
C3 plays a central role in the activation of the complement system. Its processing by C3 convertase is the central reaction in both classical and alternative complement pathways. After activation C3b can bind covalently, via its reactive thioester, to cell surface carbohydrates or immune aggregates. Derived from proteolytic degradation of complement C3, C3a anaphylatoxin is a mediator of local inflammatory process. In chronic inflammation, acts as a chemoattractant for neutrophils (By similarity). It induces the contraction of smooth muscle, increases vascular permeability and causes histamine release from mast cells and basophilic leukocytes. C3-beta-c: Acts as a chemoattractant for neutrophils in chronic inflammation. Acylation stimulating protein: adipogenic hormone that stimulates triglyceride (TG) synthesis and glucose transport in adipocytes, regulating fat storage and playing a role in postprandial TG clearance. Appears to stimulate TG synthesis via activation of the PLC, MAPK and AKT signaling pathways. Ligand for C5AR2. Promotes the phosphorylation, ARRB2-mediated internalization and recycling of C5AR2 (PubMed:8376604, PubMed:2909530, PubMed:9059512, PubMed:10432298, PubMed:15833747, PubMed:16333141, PubMed:19615750).
Plasma. The acylation stimulating protein (ASP) is expressed in adipocytes and released into the plasma during both the fasting and postprandial periods.
Involvement in disease
Complement component 3 deficiency Macular degeneration, age-related, 9 Hemolytic uremic syndrome atypical 5 Increased levels of C3 and its cleavage product ASP, are associated with obesity, diabetes and coronary heart disease. Short-term endurance training reduces baseline ASP levels and subsequently fat storage.
C3b is rapidly split in two positions by factor I and a cofactor to form iC3b (inactivated C3b) and C3f which is released. Then iC3b is slowly cleaved (possibly by factor I) to form C3c (beta chain + alpha' chain fragment 1 + alpha' chain fragment 2), C3dg and C3f. Other proteases produce other fragments such as C3d or C3g. C3a is further processed by carboxypeptidases to release the C-terminal arginine residue generating the acylation stimulating protein (ASP). Levels of ASP are increased in adipocytes in the postprandial period and by insulin and dietary chylomicrons. Phosphorylated by FAM20C in the extracellular medium.
IHC image of ab48581 staining in mouse liver formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab48581, 10µg/ml, for 15 mins at room temperature. A goat anti-chicken biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.