Thylakoid membrane protein Extraction from Arabidopsis

Protocol from CO-EXPRESSED WITH PSI ASSEMBLY1 (CEPA1) is a photosystem I assembly factor in Arabidopsis. David Rolo, Omar Sandoval-Ibáñez, Wolfram Thiele, Mark A Schöttler, Ines Gerlach, Reimo Zoschke, Joram Schwartzmann, Etienne H Meyer, Ralph Bock. Plant Cell. 2024 Oct 3; 36(10):4179-4211.doi: 10.1093/plcell/koae042.

 

Uncovering the photosystem I assembly pathway in land plants. Aihong Zhang, Lin Tian, Tong Zhu, Mengyu Li, Mengwei Sun, Ying Fang, Yi Zhang, Congming Lu. Nat Plants. 2024 Apr; 10 (4):645-660. doi: 10.1038/s41477-024-01658-3. Epub 2024 Mar 19.

 

RESISTANCE TO PHYTOPHTHORA1 promotes cytochrome b559 formation during early photosystem II biogenesis in Arabidopsis. Li-Ping Che, Junxiang Ruan, Qiang Xin, Lin Zhang, Fudan Gao, Lujuan Cai, Jianing Zhang, Shiwei Chen, Hui Zhang, Jean-David Rochaix, Lianwei Peng. Plant Cell. 2024 Oct 3; 36(10):4143-4167.doi: 10.1093/plcell/koae196.

Flow Sheet:

 

 

Procedure:

1. Collect 1g of Arabidopsis leaves, add them to 20ml Medicine I+ BSA+ ASA solution, and crush them with a homogenizer;
2. Filter the above liquid through filter membranes, then centrifuge at 4,200g at 4 ℃ for 5 minutes;
3. Discard the supernatant, suspend the precipitate in 1ml of Medium II, then centrifuge at 8,000g at 4 ℃ for 2 minutes;
4. Discard the supernatant, suspend the precipitate in 1ml Medium II-sorbitol, centrifuge at 8,000g at 4 ℃ for 2 minutes;
5. Completely suspend the precipitate in 200ul Medium II-sorbitol to obtain the thylakoid membrane protein;
6. Measure the concentration : Acetone method quantification;
7. Protein denaturation: Take 100ul of the above sample, centrifuge 12,000g at 4 ℃ for 2 minutes; discard the supernatant, add 100ul of 2 X SDS to the precipitate, and denature at room temperature (25 ℃) for 30 minutes; centrifuge 12,000g at 4 ℃ for 2 minutes, take the supernatant, and add 100ul Medium II-sorbitol.

 

Solutions Required

 

Medium I                          

0.33M  Sorbitol (M=182.17)                           60.12g

30mM  Tricine/KOH  (0.5M, pH 8.4)             40ml

5 mM  EDTA  (0.25M, pH 8.35)                       20ml

10 mM  NaHCO3  (M=84.01)                          20ml

                                                                                             

                                                        Add ddH2O to 1L

 

Medium II+sorbitol                       

0.3M  Sorbitol (M=182.17)                             13.66g

20mM  Hepes/KOH  (1M, pH 7.6)                    5ml

2.5 mM  EDTA  (0.5M, pH 8.0)                      1.25ml

5 mM  MgCl2  (M=203.3)                            0.255g

                                                                                             

                                               Add ddH2O to 250ml

 

Medium II-sorbitol                 

20mM  Hepes/KOH  (1M, pH 7.6)                    5ml

2.5 mM  EDTA  (0.5M, pH 8.0)                      1.25ml

5 mM  MgCl2  (M=203.3)                            0.255g

                                                                                             

                                               Add ddH2O to 100ml

 

Medium I+ASA+BSA        

1.87M    Ascorbate                                        20ul

10%      BSA                                                   200ul

Medium I                                                      20ml