Chin Bull Bot ›› 2020, Vol. 55 ›› Issue (1): 76-82.doi: 10.11983/CBB19208

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Protocols for Analyzing Plant Phospho-proteins

Zhu Dan,Cao Hanwei,Li Yuan,Ren Dongtao()   

  1. State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China
  • Received:2019-10-24 Accepted:2019-12-31 Online:2020-01-03 Published:2020-01-01
  • Contact: Ren Dongtao E-mail:ren@cau.edu.cn

Abstract:

Protein phosphorylation is one of the important protein posttranslational modifications that is involved in the regulation of most cellular processes in plants. Protein kinases catalyze the phosphorylation by transferring the phosphate group in ATP to the substrate proteins. The phosphate is usually covalently linked to the hydroxyl group of specific amino acid residues in the substrates by an ester bond. The mostly studied phosphorylation sites are serine, threonine, and tyrosine residues. Here, we present protocols and related tips for the in vitro and in vivo protein phosphorylation assays.

Key words: protein phosphorylation, protein kinase, plant

Figure 1

An in vitro phosphorylation assay of 3 putative substrate proteins by MPK6 in Arabidopsis thaliana MPK6 was fused with the 6×His tag and the 3 putative substrates were fused with the Flag tag. After phosphorylation reaction, the proteins in the mixture were separated on a SDS- PAGE gel. The gel was dried and exposed to X-ray film (top). Immunoblotting with anti-His and anti-Flag antibodies were used to show the levels of the substrate (middle) and kinase (bottom) proteins in the reactions."

Figure 2

Immunoblotting detection of phospho-proteins in samples after Phos-tag gel and SDS-PAGE gel separation Phospho-proteins can be dephosphorylated by phosphatases (e.g. Calf intestine alkaline phosphatase, CIAP). Protein samples treated with (+) or without (-) CIAP were separated by Phos-tag (top) and SDS-PAGE (bottom) gels, and the specific protein was further detected by immunoblotting. The P-form and None-P-form of the protein were separated clearly by a Phos-tag gel (top), while the two forms were not separated by a SDS-PAGE gel (bottom). The missing of the upper band and the increasing of the bottom band after Phos-tag gel separation indicated that P-form protein was completely dephosphorylated by CIAP treatment."

Figure 3

Pro-Q staining assay of the phospho-proteins in total proteins extracted from Arabidopsis thaliana seedlings and separated by a 2D gel Two-week-old Arabidopsis thaliana seedlings were used for total protein extraction. The total proteins were separated by a 2D gel and the phospho-proteins were stained by Pro-Q (left). The gel was scanned with a Typhoon 9410 fluorescence scanner, and then stained with blue-silver to visualize the proteins (right)."

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