Plant Science

Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His₆-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and specificity of our methods allow for purification of proteins expressed at native levels from endogenous promoters to enable uncovering the functional roles of plant protein complexes.

Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining the localization of Chlamydomonas proteins using this method are published. Here, we introduce a protocol of indirect immunofluorescence assay adapted from our papers reporting LCIB (CO₂-recycling factor in the vicinity of pyrenoid; Yamano et al., 2010), LCI1 (plasma membrane-localized inorganic carbon transporter; Ohnishi et al., 2010), HLA3 (plasma membrane-localized ABC-type bicarbonate transporter; Yamano et al., 2015), and LCIA (chloroplast envelope anion channel; Yamano et al., 2015) in Chlamydomonas reinhardtii. The protocol described here could be useful for observing the protein of interest in other algae cells.

Protein phosphorylation is one of the most common post-translational modifications in eukaryotic cells and plays a critical role in a vast array of cellular processes. Efficient methods of protein extraction and phosphopeptide purification are required to ensure the detection of high quality of proteins. In our hands, phenol extraction of proteins and TiO₂ chromatography enrich phosphorylated peptides more efficiently than other methods in the moss Physcomitrlla patens (P. patens).

In organello protein synthesis method allows the analysis of mitochondrial translation products. The principle of this method relies on incubation of isolated intact mitochondria with radiolabeled amino acids such as ³⁵S methionine. After protein synthesis, the radiolabeled translation products are subsequently separated by SDS polyacrylamide gel electrophoresis and analysed by autoradiography. For in organello analysis of protein synthesis, the isolated intact mitochondria must retain their bioenergetics capacity, and in consequence be fully functional and able to perform coupled respiration. This in turn requires a quick and gentle purification of mitochondria during their isolation.

³⁵S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of radioactive sulfur. This example uses Chlamydomonas cells and H₂³⁵SO₄ (Schwarz et al., 2012).