Dear colleagues,
I would like to seek your kind advice on click chemistry enrichment of azidohomoalanine (AGA)-tagged proteins.
From literature, I learned that there are 2 main avenues, either (1) copper-free, DBCO-agarose bead pull down method or (2) Click-it enrichment kit (e.g. Thermo C10416) based on copper catalysis and alkyne-beads covalent capture.
Ref 1 (DBCO): https://www.ncbi.nlm.nih.gov/pubmed/28104718
Ref 2 (Click-it): https://www.ncbi.nlm.nih.gov/pubmed/28928394
From your experience, which method would provide the greater enrichment efficiency without intensive optimization?
As a newbie, it may feel safer to consider using an enrichment kit (e.g. the Thermo Click-it kit), but the manufacturer's protocol recommends an input of 5–20 mg proteins. Any experience on scaling it down (e.g. using 100-200 ug protein input)?
Any feedback would be much appreciated.
Thank you,
Kay
Alkyne-based methods would be a better choice according the following procedure
Article Dual-Functional-Tag-Facilitated Protein Labeling and Immobilization
you can get a view of other DBCO based conjugation from this link
https://www.youtube.com/watch?v=SJEulFA2m5c&list=PLTCBtzqkKa4QhazLdinhoIuIx-HV6YDCX
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