Hello. I would appreciate if anyone can help me.
From the yeast 2 hybrid results, the protein of my interest seems to interact with structural proteins in muscle and heart. Because there is no cell line currently avalilable that express structural proteins abundantly, I am planning to clone my gene into a vector with histidine tag and use Pierce’s product “Pull-Down PolyHis Protein:Protein Interaction Kit (Catalog Number 21277)”. Here is what I am planning:
Bait: clone in the gene of my interest into pcDNA(tm) 3.1/V5-His (Invitrogen);, which attaches 6x His and V5 at the C-terminus, and overexpress it in HEK293T cells.
Prey: skeletal muscle from the thigh of a mouse. (tissue extract)
Does anyone try such an approach? Or any other approach?
Thank you very much for your help in advance.