Comparison of methods for extracting thylakoid membranes of Arabidopsis plants.

Abstract:

:Robust and reproducible methods for extracting thylakoid membranes are required for the analysis of photosynthetic processes in higher plants such as Arabidopsis. Here, we compare three methods for thylakoid extraction using two different buffers. Method I involves homogenizing the plant material with a metal/glass blender; method II involves manually grinding the plant material in ice-cold grinding buffer with a mortar and method III entails snap-freezing followed by manual grinding with a mortar, after which the frozen powder is thawed in isolation buffer. Thylakoid membrane samples extracted using each method were analyzed with respect to protein and chlorophyll content, yields relative to starting material, oxygen-evolving activity, protein complex content and phosphorylation. We also examined how the use of fresh and frozen thylakoid material affected the extracts' contents of protein complexes. The use of different extraction buffers did not significantly alter the protein content of the extracts in any case. Method I yielded thylakoid membranes with the highest purity and oxygen-evolving activity. Method III used low amounts of starting material and was capable of capturing rapid phosphorylation changes in the sample at the cost of higher levels of contamination. Method II yielded thylakoid membrane extracts with properties intermediate between those obtained with the other two methods. Finally, frozen and freshly isolated thylakoid membranes performed identically in blue native-polyacrylamide gel electrophoresis experiments conducted in order to separate multimeric protein supracomplexes.

journal_name

Physiol Plant

journal_title

Physiologia plantarum

authors

Chen YE,Yuan S,Schröder WP

doi

10.1111/ppl.12384

subject

Has Abstract

pub_date

2016-01-01 00:00:00

pages

3-12

issue

1

eissn

0031-9317

issn

1399-3054

journal_volume

156

pub_type

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