Week 10 HW: Imaging and Measurement

For your final project:

- Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc.

- Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.

- What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

- Toehold switch activation:

• Does the switch open specifically in response to LncRNA H19? I would order the toehold switch construct from Twist and it will be expressed at Ginkgo using a PURExpress cell-free reaction with and without the H19 trigger, using sfGFP as reporter.

Technology: fluorescence spectroscopy with a plate reader.

- Fusion protein identity:

• Was the anti-STAT3 monobody fused to the E3 ligase recruitment domain expressed at the right size and correct sequence?

Technology:

• SDS-PAGE: approximate size (first check)
• Intact LC-MS: confirm molecular weight
• Peptide mapping for LC-MS/MS: confirm the correct order of amino acids (primary structure)

- Ternary complex formation:

• Does the bioPROTAC bridge STAT3 to the cell´s degradation machinery?

Technology: native mass spectrometry I would look for three peaks:

• STAT3 (alone)
• E3 recruitment domain alone
• Assembled ternary complex (If the third peak appears, the mechanism is functional)

- LNP formulation integrity:

• Are the lipid nanoparticles used for intraperitoneal delivery correctly assembled and homogeneous?

Technology: CDMS

- STAT3 degradation:

• Do STAT3 protein levels decrease?

Technology: Western Blot in endometriosis cells treated vs. untreated.

- IL-6 and IL-8:

• Do downstream inflammatory markers (IL-6 & IL-8) drop as functional output?

Technology: ELISA