TY  - JOUR
AU  - Tan, Hua
AU  - Bungert-Plümke, Stefanie
AU  - Kortzak, Daniel
AU  - Fahlke, Christoph
AU  - Stölting, Gabriel
TI  - Dual-color Colocalization in Single-molecule Localization Microscopy to Determine the Oligomeric State of Proteins in the Plasma Membrane
JO  - Bio-protocol
VL  - 13
IS  - 13
SN  - 2331-8325
CY  - Sunnyvale, CA
PB  - bio-protocol.org
M1  - FZJ-2023-02769
SP  - 4749
PY  - 2023
N1  - Development of this protocol was supported by the Deutsche Forschungsgemeinschaft (FA 301/15-1 to Ch.F.) as part of Research Unit FOR 5046, project P4. We acknowledge the original research paper (Tan et al., 2022), from which this protocol is derived, as the primary reference when additional information is needed.
AB  - Determining the oligomeric state of membrane proteins is critical for understanding their function. However, traditional ex situ methods like clear native gel electrophoresis can disrupt protein subunit interactions during sample preparation. In situ methods such as stepwise photobleaching have limitations due to high expression levels and limitations of optical resolution in microscopy. Super-resolution microscopy techniques such as single-molecule localization microscopy (SMLM) have the potential to overcome these limitations, but the stochastic nature of signals can lead to miscounting due to over-expression, background noise, and temporal separation of signals. Additionally, this technique has limited application due to the limited selection of fluorescent labels and the demanding control of laser power. To address these issues, we developed a dual color colocalization (DCC) strategy that offers higher tolerance to background noise and simplifies data acquisition and processing for high-throughput and reliable counting. The DCC strategy was used to determine the oligomeric states of membrane proteins of the SLC17 and SLC26 family with SMLM, providing a robust and efficient method for studying protein interactions.
LB  - PUB:(DE-HGF)16
C6  - 37456335
UR  - <Go to ISI:>//WOS:001089325800011
DO  - DOI:10.21769/BioProtoc.4749
UR  - https://juser.fz-juelich.de/record/1009340
ER  -