Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. To minimize cross-reactivity, the goat anti-chicken IgY whole antibodies have been affinity-purified and cross-adsorbed against chicken serum containing non-immunoglobulin chicken serum proteins. The images were captured at 60X magnification. Nonspecific staining was not observed with secondary antibody alone (panel e). Nuclei (Panel b: blue) were stained with Hoechst33342 (Product # H1399). Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32931, 1:2000 dilution) in 0.1% BSA in PBS for 45 minutes at room temperature, was used for detection of Vimentin in the cytoplasm (Panel a: green). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 2% BSA for 1 hour and labeled with primary antibody (1:200 dilution in 0.1% BSA) for 3 hours at room temperature. The use of insolubilized adsorption antigens prevents the presence of excess adsorbent protein or immune complexes in the antiserum.Immunofluorescence analysis of Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32931) was performed using MCF10A cells stained with Vimentin Polyclonal Antibody (Product # PA1-10003). Special attention is given to the removal of antibodies to common Ig/Fab. Prior to and following reconstitution store the antibody at 2-8☌ for one month or at -20☌ for longer.Īdsorption: Immunoaffinity adsorbed using insolubilized antigens as required to eliminate antibodies cross-reacting with other components of the immunoglobulin system or reacting with other serum proteins. Restore by adding 1.0 ml of sterile distilled waterĪmmonium Sulphate Precipitation and Ion Exchange Chromatography State: Lyophilized hyperimmune purified IgG fraction PBS, pH 7.2 without preservatives and foreign proteins Cross-reactivity of this conjugate has not been tested in detail. It does not react with any non-Ig protein in chicken serum, as tested by immunoelectrophoresis and double radial immunodiffusion.Ĭross-reactivity: Inter-species cross-reactivity is a normal feature of antibodies to immunoglobulins, since Ig of different species frequently share antigenic determinants. The reactivity of the antiserum is directed to the Fc subunit of the IgM molecule which expresses strict isotypic (class) specificity. Purified IgM isolated from Chicken serum.įreund’s complete adjuvant is used in the first step of the immunization procedure. When applied in any cytochemical or histochemical staining procedure or solid phase coupling technique, the optimum concentration of the IgG preparation should be established by titration before being used.ĮLISA and comparable non-precipitating antibody-binding assays: 1/500-1/5000.Īntibody titre: Precipitin titre 1/32 when tested against pooled normal chicken serum in agar-block immunodiffusion titration. Can be used as unlabelled primary or secondary reagent for indirect detection of IgM at the cellular and subcellular level by staining of appropriately treated cell and tissue substrates to prepare conjugates of the user’s own choice to prepare an insoluble immunoaffinity adsorbent or a solid phase antibody reagent by coupling to an artificial carrier and as catching antibody in non-isotopic methodology and solid phase immunochemistry.
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