Nuclear protein Extraction from Arabidopsis

Protocol from Isolation of Nuclear Proteins. Setsuko Komatsu. Methods Mol Biol. 2007:355:73-7. doi: 10.1385/1-59745-227-0:73.

Nuclear Extraction from Arabidopsis thaliana. Fang Xu and Charles Copeland. Bio-protocol. Vol 2, Iss 24, Dec 20, 2012. doi:10.21769/BioProtoc.306.

Procedure:

1. Collect fresh plant tissue and grind the tissue to a fine powder in liquid nitrogen. Add Lysis Buffer at a ratio of 1 mg/3 mL, and homogenize the mixture by gentle shaking;
2. Filter the sample through a filter membrane into a new centrifuge tube and operate on ice throughout the process. After filtration, centrifuge at 1,500 g at 4 ℃ for 10 minutes;
3. Discard the supernatant and add NRBT buffer then homogenize the mixture, and centrifuge at 1,500 g at 4 ℃ for 5 minutes;
4. Repeat step 3 four times more;
5. Discard the supernatant and add 1 ml Lysis Buffer to the pellet. Re-suspend the nuclei by pipetting;
6. Transfer the thoroughly mixed nuclear protein into a 1.5 mL centrifuge tube, with 900 μL of nuclear protein per 100 μL of 10 × protease inhibitor cocktail;
7. Measure protein concentration;
8. Denaturation: Add 5 x Laemelli Buffer to the protein sample at a ratio of 4:1, denature at 70 ℃ for 10 minutes, and store at -80℃.

 

Solutions Required

Lysis Buffer

1M   Tris-Hcl (PH=7.5)                           1mL

1M   Kcl                                                     1mL

0.5 M EDTA                                           200μL

1M  Mgcl2                                              125μL

25% Glycerol                                       12.5mL

250 mM Sucrose                                4.278g

1M DTT                                                   250μL

Protease inhibitor cocktail (Roche)

                                                                              

                                       Add ddH2O to 50mL

 

NRBT buffer

1M   Tris-Hcl (PH=7.5)                       4 mL

1M  Mgcl2                                        500 μL

25% Glycerol                                    50 mL

10% Trixon-100                                    4 mL                   

Protease inhibitor cocktail (Roche)

                                                                              

                                  Add ddH2O to 200mL

 

5×Laemelli Buffer                  

1 M Tris-Hcl (PH=6.8)                        0.875mL

Glycerol                                                 4.5 mL

SDS                                                          0.5g

0.25% Bromophenol blue                   0.5mL

2-ME                                                     1.25 mL

                                                                              

                                        Add ddH2O to 10mL