Protocol to determine oligomerization states of proteins in living cells using photon counting histogram analysis.

Determining protein oligomerization states in living cells is essential for understanding cell signaling and disease pathology, but it remains technically challenging. Here, we present a protocol for quantifying the oligomeric states of HaloTag-labeled proteins in living mammalian cells using fluorescence fluctuation spectroscopy (FFS) and photon counting histogram (PCH) analysis. Using the photoactivatable protein cryptochrome 2 as a representative model, we describe steps for cell culture, chemical transfection, cell staining, and FFS data acquisition. We then detail procedures for PCH analysis. For complete information on the generation and use of this protocol, please refer to Camp et al.1.