Probing Protein Distribution Along the Nuclear Envelope In Vivo by Using Single-Point FRAP

Determining the locations of nuclear envelope transmembrane proteins and their concentrations across the outer and inner nuclear membranes has been a challenging and time-consuming process. Typically, this required the week-long process of fixing and immunogold staining of cells prior to analysis by electron microscopy. Here, we describe a method, single-point fluorescence recovery after photobleaching (spFRAP), which is able to quickly determine the localization and distribution of nuclear membrane proteins along the double nuclear envelope membranes with a precision of 10-15 nm in a matter of 10-20 min the day after transfection.