Methods for Protein Tags

Goal

identify higher concentrations of ACE2 receptors expressed -- displaying that our engineered macrophages are expressing soluble ACE2

Thought process for immunoprecipitation tag to test method #1 in order:

1. Find ELISA provider / comes with whole kit after sequence submission -- asking if WEHI can provide
2. Considering if this step can be done in one with testing method 2 // requires a separate experiment with ACE2 specific antibodies
3. Looking into designing ACE2 specific antibodies

Goal

identify higher concentrations of ACE2 receptors expressed -- displaying that our engineered macrophages are expressing soluble ACE2

Thought process for immunoprecipitation tag to test method #1 in order:

1. His-tag: problem with random binding given common amino acid
2. Discovered that ACE2 function optimally at a pH of 6.0 - 9.0
3. HA tag: problem, immunogenicity
4. Intein tag: problem, requires special vector
5. GST tag: problem, large size
6. Considered using FACS as the primary detection method for SP1 complex
7. Reconsidering HA tag after given guidance: immunogenicity is not an issue if the macrophages themselves express the tag
8. Deciding to create two plasmids, one with His tag and the other with HA tag
9. Finding out the tags must be placed on the C terminal end given possible interruptions with signal peptidase -- problem with placing tag on ace2 in plasmid, would be easier if the tag is not at the terminal -- tag must be at terminal // plasmid altered

Met with Suzy Butcher early on for advice

• Use endotoxin free DNA prep kits
• Suggested c-myc and FLAG tag for macrophages