Production and purification of cell culture transfected Wnt ligand

Production and purification of cell culture transfected Wnt ligand

[Essence summary]

Wnt ligand protein is abundant in vitro, but it is very difficult to purify. This method uses Wnt11r protein as an example to illustrate how to use 293T cells to produce secreted Wnt11r and collect Wnt11r protein for further biochemical experiments in vitro.
[Materials and Reagents]
1. 293T cells
2. Fetal Bovine Serum
3. DMEM
4. PEN/Streptococcus
5. Trypsin of EDTA
6. PBS buffer
7. Effectene transfection reagent
8. Anti-FLAG M2
9. 0.1M Glycine-Hydrochloric Acid PH3.5
10. 0.5 M Tris-HCl

[Equipment]
1. Centrifuge
2. Water bath
3. 50ml conical tube
4. 100 mm Petri Dish

types of petri dishes,petri dish plastic 90mm sterile,petri dish petri,petri dish disposable
[Step]
1. Isolate 293T cells:
1) At room temperature, warm medium (DMEM medium + 10% fetal bovine serum with 1% PEN/Streptococcus) and PBS buffer in a 37°C water bath and 0.05% trypsin EDTA. Obtain a 50 ml conical tube and a 100 mm Petri dish to transfer the cells.
2) Aspirate the old medium. Add 10 ml of PBS buffer to rinse the cells. Shake the petri dish gently. Aspirate Hass buffer.types of petri dishes,petri dish plastic 90mm sterile,petri dish petri,petri dish disposable
3) Add 1 ml of 0.05% trypsin EDTA to a 100 mm culture dish to fuse with 293T cells. 1 minute at 37°C. Shake the petri dish gently. You will see the cells detach from the culture dish. Add 9 ml of medium and gently pipette up and down to detach the cells from the plate. After separation, pour into a 50 ml conical tube.
4) Centrifuge the cells at 1000 RPM for 3-4 minutes. Remove the supernatant with a vacuum Pasteur pipette. Add 10 ml of medium and gently pipette up and down to resuspend the cells.
5) Add 9 ml of fresh medium and 1 ml of suspended cells to a new petri dish. This petri dish will be used until the 5th day. You can also make a 1:5 and 1:20 dilution, and the cells will converge on the third and seventh days respectively.types of petri dishes,petri dish plastic 90mm sterile,petri dish petri,petri dish disposable
6) The cells are grown in a 37°C, 5% CO2 incubator. Check the medium after 3 days, if the medium turns light yellow, change the medium.

2. Inoculation of cells and transfection:
7) Dilute the cells like steps1-6 in a 100 mm Petri dish to a ratio of 1:10. Grow for 16-24 hours. These cells will become 30-40% joint.
8) Warm medium and Hass buffer at 37°C. Mix the DNA in the lid (Wnt11r expresses a FALG logo). Follow the transfection steps from Qiagen:
a. Mix 8 μg of DNA in 300 μl EC buffer.
b. Add 64μl enhancer, mix well and pipette. Incubate under the lid for 5 minutes.
c. Add 60μl effectene, mix well, and incubate for 10 minutes.
d. Add 3000μl of medium to the mixture.
Note: The above amount is for transfection in a 100 mm Petri dish.
e. During the incubation period, remove the old medium from the cells and rinse once in 10 ml PBS buffer.
f. Add 7 ml of fresh medium to the cells. When the incubation is over, add the mixture to the petri dish (3424 μl in total: 300 μl+64 μl+60 μl+3000 μl). Shake the petri dish gently.types of petri dishes,petri dish plastic 90mm sterile,petri dish petri,petri dish disposable
g. Cells in 37/CO2 incubator.
9) Day 2: Remove the transfection medium from the petri dish. Add another 10 ml of fresh medium (slowly pipette the medium along the edge. The cells are expelled except for the medium through the pipette). Grow for 3-4 days.
Note: This step is completely optional. The cells grow well in the effectene transfection medium.

3. Collect Wnt11r-FLAG supernatant:
10) After 4 days of growth in a 15 ml conical tube. Spin at 1000RPM for 5 minutes. Filter through nitrocellulose membrane (0.2 micron). According to the literature, Wnt signaling ligand can be stored at 4°C for several months without loss of activity. But I don’t know if wnt11r is also. Optional: Dispense 1 ml of supernatant and store at -80°C.
11) Pull down the wnt11r marker protein with anti-FLAG beads.
a. Thoroughly suspend the anti-FLAG M2 gel in the vial. The ratio of suspension to gel is 2:1. (That is, if you want to get 20μl of beads, use a wide-tip pipette to remove 40μl from the vial). Follow the manufacturer’s instructions to clean the anti-FLAG beads.
b. Wash the beads with cold TBS buffer (40μl gel suspension 500μl) 4 times. Centrifuge the beads at 5000 – 8000g for 30 seconds. Wait 1-2 minutes before processing the sample. Remove the supernatant with the tip of a pipette. Be careful not to transfer any beads.
c. Wash the beads with 0.1 M glycine-hydrochloric acid pH 3.5 to reduce unbound antibodies.
d. Note: Do not put the beads in glycine hydrochloric acid for more than 20 minutes.
e. Wash the beads again with TBS buffer for a total of 3 times.
f. Add wnt11r medium to the beads. Tube placed on the wheel 4 °C = O / N
Optional: You can add protease inhibitors to the medium before adding them to the beads.
Note: Each of the above steps should be on ice or at 4 °C.
12) Use glycine-hydrochloric acid buffer to elute the wnt11r-FLAG protein.
a. Centrifuge the beads and remove the medium with a pipette tip.
b. Wash the magnetic beads 3 times with TBS buffer. Please make sure that all the supernatant is discarded.
c. Elute the wnt11r-FLAG protein with 0.1M glycine-hydrochloric acid.
d. Add 100ul 0.1M glycine-HCl buffer to the beads. The incubated samples are gently shaken at room temperature for 5 minutes.
e. Centrifuge at 5000-8000g for 30 seconds.
f. Transfer the supernatant to a fresh tube containing 10 μl 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl. Be careful not to transfer any beads.
g. The supernatant can be used immediately when stored at 4 °C. Store at -20 °C for long-term storage.
13) Determine the amount of protein in the elution buffer.
a. Elute the protein in 10μl and add 10μl of 2XSDS sample buffer. Boil for 5 minutes, then run the SDS-PAGE gel. For the markers, different amounts of bovine serum albumin were run in each lane. Stain the gel with G-blue, and then determine the amount of protein in 10 μl of eluate.
b. The size of wnt11r-FLAG is 44KD.