Figure 2.

Qualitative analysis of scFv molecules in different lysate fractions using autoradiography. Cell-free synthesis of scFvs was performed in presence of ¹⁴C-leucine using the insect cell-free expression system. Immediately following the protein synthesis reaction, translation mixtures (TM) were centrifuged and the supernatant fraction (SN1) was separated from the vesicular fraction (VF1). VF1 was resuspended in PBS supplemented with 0.2% DDM. After an additional centrifugation step, SN2 was separated from the remaining VF2, resulting in altogether five lysate fractions that were subsequently analyzed by SDS-PAGE and autoradiography. (A) Detection of scFv molecules without signal sequence (SH527-IIA4, SH527-IIC10, SH855-C11) in TM, SN1, and VF1. (B) Detection of scFv molecules with melittin signal sequence (Mel-SH527-IIA4, Mel-SH527-IIC10, Mel-SH855-C11) in TM, SN1, VF1, SN2, and VF2. Asterisks indicate lysate fractions that were directly used for functional analysis of scFv antibody fragments by ELISA.