Fab cloning, expression, purification and validation

PF Peiqiang Feng
XW Xudong Wu
SE Satchal K. Erramilli
JP Joao A. Paulo
PK Pawel Knejski
SG Steven P. Gygi
AK Anthony A. Kossiakoff
TR Tom A. Rapoport
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Specific binders based on phage ELISA results were sequenced at the University of Chicago Comprehensive Cancer Center DNA Sequencing facility and unique clones were then subcloned into the Fab expression vector RH2.2 (gift of S. Sidhu) using the In-Fusion Cloning kit (Takara). Successful cloning was verified by DNA sequencing. Fabs were then expressed and purified as previously described35. Following purification, Fab samples were verified for purity by 4–20% SDS–PAGE and subsequently dialysed overnight in 25 mM HEPES pH 7.4 and 150 mM NaCl. Purified Fab affinities were estimated by multi-point ELISA42 using the ligase complex in biotinylated nanodiscs.

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