Product is the lyophilized powder of fluorescein-5-isothiocyanate (FITC "Isomer I")-conjugated goat IgG fraction to guinea pig complement C3 and buffer salts.
Background Information
The complement system provides innate defense against microbial infection and is a "complement" to antibody mediated immunity. The complement system consists of thirty-five interacting plasma and membrane associated proteins which contribute to host-defense and initiate and amplify inflammation, even in the preimmune state where specific antibodies and lymphocytes are not available. In addition to the complement components themselves, this system also contains several soluble factors that prevent spontaneous complement activation from occurring in solution, as well as, several regulatory membrane associated proteins that protect host cells from accidental complement attack. Cleavage of the C3 component by either the classical pathway or the alternative pathway releases C3a and C3b. C3a appears to be important in many inflammatory responses while the C3b fragment covalently binds to the cell or bacterial surface and plays a role in opsonisation. Binding of C3b to the C4b component of the C3 convertase, results in C5 convertase (C4b3b2a) formation.
Applications
Fluorescein-Conjugated Goat IgG Fraction to Guinea Pig Complement C3 is suitable for use as a reagent in immunofluorescence assays (IFA), cell staining (fluorescent microscopy and cell sorting), tissue staining, and blot immunostaining. This product is especially useful in anti-complement immunofluorescent (ACIF) assays.
Recommended Use
Reconstitute product with 2.0 mL of deionized or distilled water. Gentle swirling may be used to speed rehydration. Avoid vigorous shaking of the reconstituted material.
Typical Working Concentration
Suggested working dilution: Anti-complement immunofluorescent (ACIF) assays: 1:15
Preparation Method
Antibody and FITC are conjugated under defined conditions to obtain the specified molar ratio (3-6 mol/mol). Conjugated protein is purified by gel filtration. The product is dialyzed into 0.02M sodium phosphate, 0.14M sodium chloride, pH 7.3, with 0.05% sodium azide, adjusted to standard titer, filtered through a 0.22 µm filter, vialed, and lyophilized.
Analysis Note
The total protein is measured using the Biuret procedure with bovine albumin as the standard; F/P ratio is calculated using absorbance at 491.5nm. Antibody titer of the IgG fraction is standardized with an in-house control by immunoelectrophoresis. The IgG fraction is tested for purity and specificity at 40 mg/mL using immunoelectrophoresis; No trace of albumin is detected. Antibody activity to serum albumin is not present.