Analysis Note
The total protein is measured by absorbance (A280nm); F/P ratio is calculated using absorbance at 491.5nm. Antibody titer of the affinity purified F(ab')2 fragments is standardized by equivalence-point precipitation. The affinity purified F(ab')2 fragment is tested for purity at 10 mg/mL using immunoelectrophoresis.
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 and 10% glycerol (v/v), adjusted to standard titer, filtered through a 0.22 µm filter, and vialed.
Background Information
Human IgGs are glycoprotein antibodies that contain two equivalent light chains and a pair of identical heavy chains. IgGs have four distinct isoforms, ranging from IgG1 to IgG4. These antibodies regulate immunological responses to allergy and pathogenic infections. IgGs have also been implicated in complement fixation and autoimmune disorders. Specificity of anti-human IgG (whole molecule)-FITC antibody for human IgG is determined by immunoelectrophoresis against purified human IgA, IgG, IgM, and Bence Jones κ and λ myeloma proteins.
Applications
Fluorescein-Conjugated Goat Affinity Purified F(ab')2 Fragment to Human IgG (Whole Molecule) is suitable for use in direct immunofluorescence, as well as for ANA (Anti-Nuclear Antibody)-indirect immunofluorescence. The antibody may also be used in FACS-based assays. It is also suitable for use as a reagent in immunofluorescence assays (IFA), cell staining (fluorescent microscopy and cell sorting), immunostaining of acetone-fixed frozen and formalin-fixed, paraffin-embedded tissue sections, and blot immunostaining. Use of affinity purified antibodies is recommended to avoid non-specific binding from the inherent antibodies of host animals; these F(ab')2 fragments are recommended for staining of cells or tissues which contain Fc receptors.
Product Description
Fluorescein-Conjugated Goat Affinity Purified F(ab')2 Fragment to Human IgG (Whole Molecule) is fluorescein-5-isothiocyanate (FITC "Isomer I")-conjugated goat affinity purified F(ab')2 fragments to human IgG (whole molecule) and buffer salts.
Typical Working Concentration
ANA (Anti-Nuclear Antibody)-indirect immunofluorescence: 1:32; Direct immunofluorescence: 1:16. Dilution recommended: 1:100 - 1:400 for Tissue Staining; 1:250 - 1:1,000 for Immuno Blotting; 1:1,000 - 1:4,000 for ELISA/FIA.