Antibody Detection

Part IIPart II

Detection of AntigensWestern Blotting “Dot-Blot Assay”

Positive Control

Primary antibody (IgY)

LysedE. Coli Cells

Lysed Salmonella Cells

Nitrocellulose MembraneHas a high affinity to all proteins

1 Binding Ag (antigen) onto membrane. (E. Coli & Salmonella)

Pre-lab & Lab Protocol #1

2. Blocking with 3% BSA. In 1% PBS (Phosphate buffer solution)

Lab Protocol #2 - 3

Mrs. Bielic say’sMrs. Bielic say’s::BSA is a Large inert’t’t’ BSA is a Large inert’t’t’

Protein moleculeProtein molecule

STOPSTOPSWITCH SWITCH TO PART ITO PART I

3. Application of Primary Antibody (IgY)Your Antibodies you isolated form chicken egg

Lab Protocol: #4

4. Rinse non-specifically bound Primary Antibodies with PSB (Phosphate buffer solution)

Lab Protocol # 5 - 7

5. Application of Secondary antibody(HRP-Horseradish Peroxidase Conjugated, goat anti – IgY)

Lab Protocol # 8 - 9

6. Rinse non-specifically bound Secondary Antibodies with PSB

(Phosphate buffer solution)Lab Protocol # 10 -12

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

BSASalmonellaE. coli

Primary Ab

Secondary Ab

TMB

BSA