Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

Abstract GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused itself may occasionally affect the and/or stability of targeted protein, leading to both false-positive and false-negative results in Furthermore, fusion technology is not well suited some proteins, depending on their topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, which proteins are a small peptide tag recombinantly expressed. The whole-cell extracts solubilized, mixed with anti-peptide Nb analysis. FSEC-Nb enables evaluation expression, monodispersity thermostability without need purification but does require direct proteins. Our show as powerful tool screening structural functional studies.