Part 3 - Video Lecture Notes
Thursday, February 7, 2019 10:13 PM
Antibody-based Techniques
• Western Blots (Immunoblot):
○ Antibodies used to bind to protein of interest.
○ Add a fluorescent tag to visualize antibody.
1. Transfer proteins from an SDS-polyacrylamide gel to a polymer sheet.
a. Must transfer to polymer sheet: antibodies cannot penetrate gel.
2. Add the fluorescently labeled antibody.
3. "Wash" to remove any unbound antibody. Add solvent and shake.
4. Detect fluorescence on polymer sheet with bound antibodies.
a. The protein band detected is the protein of interest.
• Immunoprecipitation:
○ Antibodies bound to beads will bind to estrogen (protein of interest) receptors once mixed.
1. Centrifuge the soln. All the antibody-bound estrogen will drop to the bottom.
2. Discard supernatant. Keep pellet w/ estrogen.
3. Wash out proteins stuck b/w antibody-estrogen complexes.
4. Add denaturant. This will release binding of estrogen and antibody.
5. Centrifuge again. Antibody beads will drop to the bottom, and estrogen will stay at the top.
6. Discard pellet. Keep supernatant w/ purified estrogen.
Thursday, February 7, 2019 10:13 PM
Antibody-based Techniques
• Western Blots (Immunoblot):
○ Antibodies used to bind to protein of interest.
○ Add a fluorescent tag to visualize antibody.
1. Transfer proteins from an SDS-polyacrylamide gel to a polymer sheet.
a. Must transfer to polymer sheet: antibodies cannot penetrate gel.
2. Add the fluorescently labeled antibody.
3. "Wash" to remove any unbound antibody. Add solvent and shake.
4. Detect fluorescence on polymer sheet with bound antibodies.
a. The protein band detected is the protein of interest.
• Immunoprecipitation:
○ Antibodies bound to beads will bind to estrogen (protein of interest) receptors once mixed.
1. Centrifuge the soln. All the antibody-bound estrogen will drop to the bottom.
2. Discard supernatant. Keep pellet w/ estrogen.
3. Wash out proteins stuck b/w antibody-estrogen complexes.
4. Add denaturant. This will release binding of estrogen and antibody.
5. Centrifuge again. Antibody beads will drop to the bottom, and estrogen will stay at the top.
6. Discard pellet. Keep supernatant w/ purified estrogen.
