Specific cellular labeling of nuclear and membrane proteins with antibody alternatives: Affimer and nanobody

Antibodies have proven to be useful tools in fluorescence imaging to identify targets in cells. In large part, this is because they have specific immunoreactivity to their targets. However, they also have serious problems: cross-reactivity with non-target proteins, poor cell permeability, batch-to-batch variability, complicated production procedures, and large size (~150 kD) that makes them impermeant to the cell membrane and require onerous efforts for live cell labeling. Hence, we explore alternative binding proteins. We chose three antibody-like proteins which are all small (< 15 kD), yet have very high affinity and specificity to their targets and, if necessary, can be easily driven through the cell and nuclear membrane. For internal labeling, we use an Affimer to detect the Y537S mutant of the Estrogen Receptor alpha, and a nanobody to detect tumor suppressor protein p53, two important proteins involved in cancer. We also use a commercially available nanobody, called Spot nanobody, to detect the AMPAR and N-methyl-D-aspartate receptor (NMDAR), two important cell membrane-bound receptor proteins in the neuron synapse. In all cases, we conjugate these antibody-like proteins with organic fluorophores and deliver them into live cells by transiently permeabilizing the cells with Streptolysin O (SLO). Single molecule sensitivity is shown for each of them and important biological results are presented.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste