![]() ![]() ![]() 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 anti-chicken secondary antibody (Product # A32759) specifically detects the chicken primary antibody and not the mouse and rabbit primary antibodies. Fluorescent detection was performed usingiBrightFL1500 (Product # A44115). Secondary antibodies (Product # A32759), (Product # A32808) and (Product # A48269) were used for detection of Vimentin, Cytokeratin 5 and alpha tubulin respectively. The blot was probed with Vimentin Chicken IgY Polyclonal Antibody (Product # PA1-10003), Cytokeratin 5 Rabbit Polyclonal Antibody (Product # PA5-32465) and alpha Tubulin Monoclonal Antibody (YL1/2) (Product # MA1-80017). Resolved proteins were transferred onto anitrocellulose membrane (Product # IB23001) byiBlot® 2 Dry BlottingSystem (Product # IB21001). Whole cell extracts of MCF 10A (Lane 1, 2), HeLa (3, 4), SH-SY5Y (Lane 5, 6, 7) and MCF7 (Lane 8) were electrophoresed usingNuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP03222BOX). ![]() Multiplexed fluorescent western blot was performed using Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Product # A32759). Silver staining was performed to establish equivalent loading of purified proteins using the Pierce™ Silver Stain Kit (Product # 24612) (Fig. The blot was probed with Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Product # A32759, 1:2,000 dilution) and detected using theiBrightFL1500 (Product # A44115). Resolved proteins were then transferred onto a nitrocellulose membrane(Product # IB23001) byiBlot® 2 Dry Blotting System (Product # IB21001). a) wereelectrophoresed usingNuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Purified protein (100 ng) of Mouse IgG (Lane 1), Mouse IgM (Lane 2), Rabbit IgG (Lane 3), Rat IgG (Lane 4), Chicken IgY(Lane 5), Goat IgG (Lane 6),Human IgG (Lane 7), Mouse IgG1 (Lane 8), Mouse IgG2a (Lane 9), Mouse IgG2b (Lane 10), Mouse IgG3 (Lane 11) (Fig. The images were captured at 60X magnification.įluorescent western blot was performed using Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Product # A32759) and ~68 kDa band corresponding to Chicken IgY Heavy was observed in Chicken IgY but not Mouse IgG, Mouse IgM, Rabbit IgG, Rat IgG, Goat IgG, Human IgG, Mouse IgG1, Mouse IgG2a, Mouse IgG2b, Mouse IgG3. Nonspecific staining was not observed with secondary antibody alone (panel e). Nuclei (Panel b: blue) were stained with Hoechst33342 (Product # H1399) and Alexa Fluor™ 488 Phalloidin (Product # A12379, 1:300) (Panel c: green). Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Product # A32759, 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: Red. 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. Immunofluorescence analysis of Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Product # A32759) was performed using MCF10A cells stained with Vimentin Polyclonal Antibody (Product # PA1-10003). ![]()
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