Hi everyone,
I'm part of a team keen on developing a deeper understanding of protein interactions within a cellular context. Our primary goal is to identify and implement the most effective assay for measuring interactions between:
We are specifically looking for methods that:
- Function within a cellular context (not in-vitro)
- Provide quantifiable affinity scores for comparison
- Allow high-throughput analysis, potentially comparing approximately 500 proteins against a comprehensive proteome or an extensive peptide library
We are navigating through the initial stages of this project and would immensely appreciate any insights, experiences, or recommendations on methodologies, technologies, or specific challenges we should anticipate.
Thank you in advance for your valuable input!
Dear Chen Lior
I believe you can try to use the holdup method adapted to cellular context. You can gie a look to Zembo et al. Sci. Adv. 2022 "Native holdup to measure binding affinities from cell extracts". The title alone already evokes all the requests you have.
Best
Hello,
I'm not sure if this is useful to you. It seems like it could be if one of the proteins/peptides in your desired interactions were extrinsic. See the attached paper or otherwise familiarize yourself with the field of Bioorthogonal Chemistry. It isn't my field, but I understand it to be a way of chemically labeling targets within a living system such as a cell. Your extrinsic protein/peptide could be labeled with a FRET pair to the intrinsic one.
Labeling everything for FRET could decrease the throughput beyond what you would like, but if it doesn't you will need a fluorescence plate reader. To analyze cells you would use transparent plates and optics that work through the bottom of the well. I have a Tecan Infinite M200Pro that achieves this and uses double monochromators for wavelength discrimination. I suspect, but don't know, that this kind of setup would give superior results to a filter-based plate reader. Doesn't have to be Tecan of course, but I have been happy with mine.
Best of luck!
Article Bioorthogonal Chemistry and Its Applications
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