Journal Article

FZJ-2023-02769

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Dual-color Colocalization in Single-molecule Localization Microscopy to Determine the Oligomeric State of Proteins in the Plasma Membrane

Tan, H. (Corresponding author) ; Bungert-Plümke, S. ; Kortzak, D.FZJ* ; Fahlke, C.FZJ* ; Stölting, G.

2023
bio-protocol.org Sunnyvale, CA

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Please use a persistent id in citations: doi:10.21769/BioProtoc.4749  doi:10.34734/FZJ-2023-02769

Abstract: 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.

Note: 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.

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Contributing Institute(s):

1. Molekular- und Zellphysiologie (IBI-1)

Research Program(s):

1. 5241 - Molecular Information Processing in Cellular Systems (POF4-524) (POF4-524)

Appears in the scientific report 2023

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Database coverage:
; ; ; Clarivate Analytics Master Journal List ; Emerging Sources Citation Index ; IF < 5 ; JCR ; PubMed Central ; SCOPUS ; Web of Science Core Collection

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