antibody detection
Post on 31-Dec-2015
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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
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