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Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

Published: February 14th, 2019



1Department of Plant Biology, Michigan State University, 2Department of Chemistry, Michigan State University

Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Photosynthetic organisms must constantly adjust their physiology to changing environmental conditions to maximize their productivity and successfully compete with neighbors1. This is especially true of the machinery responsible for the light reactions of photosynthesis, as ambient light conditions can fluctuate by three orders of magnitude between shadows and full sunlight. Additionally, environmental factors such as drought, cold, or heat stress can reduce the availability of carbon dioxide for carbon fixation, which is the natural electron sink for the products of the light reactions. Plants must, therefore, harvest and utilize solar radiatio....

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1. Stock Solutions Preparation for Pouring Native Green Gels

  1. Prepare a 4x concentrated buffer solution for resolving gels consisting of 40% glycerol, 200 mM glycine, and 100 mM Tris buffered to pH 8.3.
  2. Prepare a 4x concentrated buffer solution for stacking gels consisting of 40% glycerol, 200 mM glycine, and 100 mM Tris buffered to pH 6.3.
  3. Store these buffers at 4 °C to prevent the growth of mold.
    Note: The buffers are stable for months at 4 °C, so preparation of 100-200 m.......

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Representative results for green gel electrophoresis are presented in Figure 1. Lane 1 provides an example of ideal results for green gel electrophoresis of spinach thylakoids, in which a maximum number of clear, sharp green bands are visible. These results are somewhat atypical, in part because not all of the bands seen in lane 1 are normally present in a given sample. Additional sample cleanup, in the form of chloroplast isolation before thylakoid solubiliz.......

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A successful thylakoid solubilization and native gel run will result in the resolution of multiple distinct visible green bands on the gel without significant distortion or smearing of the bands. Overloading the gel, a high detergent concentration, an incorrect sample pH, undissolved material, running the gel too rapidly or at too high a temperature, and an improperly poured gel are all factors that may contribute to poorly resolved thylakoid complexes. While optimizing the conditions of the gel itself (e.g., ac.......

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Funding and support were provided by the Department of Chemistry at Michigan State University.


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Name Company Catalog Number Comments
Glycine Sigma G8898
Tris base Sigma #648310
SDS Sigma L3771
Decyl Maltoside Sigma D7658 n-decyl beta d maltopyranoside, not dodecyl maltoside or alpha decyl maltoside
Octyl Glucoside Sigma O8001
Acrylamide BioRad 161-0148 37.5/1 C 40% solution
TEMED BioRad 161-0800
Ammonium Persulfate BioRad 161-0700
Magnesium Chloride Sigma M2670
Potassium Chloride Sigma P9333

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