Dual-Luciferase Transient Expression Assays of Arabidopsis Protoplasts

Preparation and transformation of protoplasts in Arabidopsis Thaliana

Day 1: Enzymatic hydrolysis

1. Prepare a piece of A4 paper, stick the double-sided tape on it. The leaf sticks on the front side of double-sided tape. Tear off the leaf epidermis with the sticky tape, then put it into the culture dish filled with enzymolysis solution. Wrap the culture dish with tin foil to against exposure to light. Put it on a shaker with 40-50r/min for enzymatic hydrolysis for 2-3 hours. (The number of leaves is about 20-30, the plants cultured for 3-5 weeks at vegetative stage. The growth conditions are as follows: grown at 23°C, 10h Light/ 14h Dark, 60-70%RH)

2. After the enzymatic hydrolysis started, prepare the PEG solution and plasmids.

3. Add equal volume of W5 solution (prepared in ice bath) into the enzymolysis protoplasts, and filter the solution and transfer it into a 50ml centrifuge tube, centrifuged at 1000rpm for 4 min at 4°C. After centrifugation, discard supernatant quickly and gently.

4. The protoplasts were resuspended with 20 ml of W5 solution on ice, centrifuged at 1000rpm for 4 min at 4°C, discard the supernatant (wash the protoplasts).

5. The protoplasts were resuspended with 30 ml of W5 solution, incubate on ice in the dark for (30~60min) (microscopic examination)

6. The samples were centrifuged at 1000rpm for 4 min at 4°C, Carefully remove the supernatant with a pipette.

7. Resuspended protoplasts with an appropriate amount of MMG solution. For each reaction: 20 µL plasmid (1 µg/ µL) + 200 µL protoplast + 220 µL PEG solution. Mix gently and incubate at room temperature (RT) for 10-15 min. 

8. Add 1 ml W5 solution to terminate the reaction.

9. Centrifuge at 100 X g for 2 min at RT, then remove 800μl supernatant, settle the sample for 10min. After centrifugation again, the remaining supernatant was removed carefully by pipette or vacuum filter. 

10. Add 1ml of WI solution to each reaction. (microscopic examination)

11. Transfer protoplasts resuspend into 2ml EP tube and incubated under light for 8-12h (shaken 2-3 times).

Day 2 (operation on ice)

12. Transfer the samples into a 1.5 ml EP tube, Centrifuge at 100 X g for 2 min at 4 °C, Discard the supernatant after centrifugation. 

13. Settle the sample for 10 minutes, centrifugation again for 1 minute, discard the supernatant.

14. Diluted 5 X Lysis Buffer (Dual-LUC Reporter Assay kit, Promega)to 1X with deionized water, add 25μl into the protoplast, Vortex for 10s and thensettle for 5 minutes, and repeated twice. (Thaw the 5 x Lysis Buffer will cost 0.5-1h on ice)

15. Centrifugation at 12000rpm for 2min at 4°C, transfer the supernatant into a new centrifuge tube and placed on ice (20μL), as the sample for subsequent experiments.

The determination of LUC

Prepare a 96-well ELISA plate.

(When opening the kit for the first time, pour 10ml ‘Luciferase Assay Buffer II Luciferase Assay’ into the brown bottle of ‘Luciferase Assay Substrate’, the solution turns pale yellow after mixing, then divide it into 1ml/ tube and wrap it with tin foil. Stored frozen at - 70°C)

(LUC substrate should be thawed avoiding light on ice for 1-2h)

1. Add above samples in 96-well ELISA plate with 5μl each, perform each assay in triplicate.

2. Add 20μl of LUC substrate every 5 seconds. 

3. LUC was measured by GloMax Navigator Microplate Luminometer (Promega)

The determination of Renilla

Mix: Stop buffer: Renilla substrate =49:1 (490 μL Stop buffer and 10 μL Renilla substrate) (Renilla substrate should be thawed for 0.5-1h on ice) (The Mix should be prepared when using)

1. Add 20 μL of the fresh Mix every 10 seconds.

2. Renilla activity was measured by GloMax Navigator Microplate Luminometer (Promega).

Solution Formulations 

1. Enzymolysis solution (stored at 4°C, use within a week) (Enzymolysis solution is light brown and clear)

Dissolve in water, heat in 55°C water bath for 10 minutes, cool to room temperature, add 200 μL 1M CaCl₂ (20mM) and 200 μL 10% BSA (0.1%), use KOH adjust pH=5.7 with KOH (0.45 μM filter sterilization) (Optional: add 352 µL β-mercutoethanol (5mM))

2.PEG solution (prepared at 1.5hour before use, dissolved by continuous rotation after vortex)

3. W5 solution 1000ml (stored at 4℃)

use KOH to adjust pH=5.7, autoclaved for 20min

4. 50ml MMG solution (stored at 4℃)

use KOH to adjust pH=5.7, autoclaved for 20min

5. WI 50 ml solution

6. 5%CS (Calf Serum)

7. Other reagents

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