This product has been prepared by immunoaffinity chromatography using immobilized antigens followed by extensive cross-adsorption against human serum proteins and collagen and non-collagen extracellular matrix proteins to remove any unwanted specificities. Typically less than 1 % cross reactivity against other extracellular matrix proteins was detected by ELISA against purified standards. This antibody reacts with most mammalian Fibronectins and has negligible cross-reactivity with Type I, II, III, IV, V or VI Collagens or Laminin. Non-specific cross reaction of anti-Fibronectin antibodies with other human serum proteins or non-Fibronectin extracellular matrix proteins is negligible.
Characteristics
Molar Ratio: 10-20 BAC molecules per Rabbit IgG molecule.
Purification
Immunoaffinity Chromatography.
Immunogen
Fibronectin was purified from Human plasma by binding to a denatured gelatin column followed by elution with high concentrations of arginine. The eluted material was further purified by gel filtration. Immunization occurred after single-band purity was assessed by SDS-PAGE.
Anti-Fibronectin antibodies have been used for indirect trapping ELISA for quantitation ofantigen in serum using a standard curve, for Immunoprecipitation and for Western blottingfor highly sensitive qualitative analysis. This product was assayed by immunoblot and found to be reactive against Fibronectin at adilution of 1: 5,000 to 1: 10,000. This product was also assayed against 1.0 ?g of Fibronectin in a standard capture ELISAusing Peroxidase Conjugated Streptavidin and ABTS(2,2'-azino-bis-[3-ethylbenthiazoline-6-sulfonic acid]) as a substrate for 30 minutes at
Restrictions
For Research Use only
Reconstitution
Restore with 0.1 mL of deionized water or equivalent.
Concentration
1.0 mg/mL (by UV absorbance at 280 nm)
Buffer
0.02 M Potassium Phosphate, 0.12 M Sodium Chloride, pH 7.2, containing 10 mg/mL BSA (IgG and Protease free) as stabilizer and 0.01 % (w/v) Sodium Azide as preservative.
Preservative
Sodium azide
Precaution of Use
This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice
Avoid repeated freezing and thawing. Dilute only prior to immediate use.
Storage
4 °C/-20 °C
Storage Comment
Store vial at 2-8 °C prior to restoration. Restore with deionized water (or equivalent), centrifuge product if not completely clear after standing at room temperature. This product is stable for one month at 2-8 °C as an undiluted liquid. For extended storage reconstitute product with 50% glycerol instead of water and then aliquot contents and freeze at -20 °C or below.
Human fibronectin has a molecular weight of 450 kDa when purified in an intact form from plasma. Fibronectin is a glycoprotein synthesized in the liver for the circulating blood plasma form, and is synthesized by many mesenchymal cells, for the extracellular matrix form. It is composed of two similar, but not identical protein chains, which are linked by two disulfide linkages at the C-terminal end of the chains. The chains are composed of domains which have specific secondary structures linked together by regions which are especially susceptible to proteolytic action. For this reason, detection by immunoblot (western) may show considerable variability in the observed apparent molecular weights, which is predicated on the source of the fibronectin, and to what degree proteolysis has occurred. Bands approximately 225 kDa shoeld be observed after SDS-PAGE when reducing and denaturing conditions are used.Synonyms: CIG, Cold-insoluble globulin, FN1