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Antibody Detection

Antibody Detection. Part II. Nitrocellulose Membrane Has a high affinity to all proteins. Lysed Salmonella Cells. Lysed E. Coli Cells. Positive Control Primary antibody (IgY). Detection of Antigens Western Blotting “Dot-Blot Assay”.

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Antibody Detection

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  1. Antibody Detection

  2. Part II

  3. Nitrocellulose Membrane Has a high affinity to all proteins Lysed Salmonella Cells Lysed E. Coli Cells Positive Control Primary antibody (IgY) Detection of Antigens Western Blotting “Dot-Blot Assay” 1 Binding Ag(antigen) onto membrane. • (E. Coli & Salmonella) • Pre-lab & Lab Protocol #1

  4. Mrs. Bielic say’s: BSA is a Large inert’t’t’ Protein molecule 2. Blocking with 3% BSA. • In 1% PBS (Phosphate buffer solution) • Lab Protocol #2 - 3

  5. STOP SWITCH TO PART I

  6. 3. Application of Primary Antibody(IgY) • Your Antibodies you isolated form chicken egg • Lab Protocol: #4

  7. 4. Rinse non-specifically bound Primary Antibodies with PSB (Phosphate buffer solution) • Lab Protocol # 5 - 7

  8. 5. Application of Secondary antibody • (HRP-Horseradish Peroxidase Conjugated, goat anti – IgY) • Lab Protocol # 8 - 9

  9. 6. Rinse non-specifically bound Secondary Antibodies with PSB • (Phosphate buffer solution) • Lab Protocol # 10 -12

  10. 7. Develop with TMB (HRP Substrate) • Lab Protocol # 13 -16

  11. TMB Secondary Ab Primary Ab Salmonella E. coli BSA BSA

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