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Transfection of HEK cells with pHY42 (melanopsin) and pGL-GFP
Protocol: Transfection of HEK cells
This is the transfection protocol used at the LBTC lab for HEK cells. The transfection reagent is PEI ([http://en.wikipedia.org/wiki/Polyethylenimine polyethylenimine]).
Please use the provided Excel sheet to calculate the volume of plasmid you should add to the cells. Replace every value in red by your own, then print the sheet out and follow the provided protocol.
The difference with CHO cells is that a different cell density is required for transfection. You also use two different mediums (one for transfection and one for growth) instead of only one.
1. Passage seed 1 day prior to transfection.
2. Prepare tubes by addition of calculated volume of DNA as a droplet at the bottom.
3. Centrifuge (1500 rpm, 3 min) the necessary volume of seed, remove conditioned medium with the pump (use a 2 ml serological pipet with a broken neck) and resuspend in necessary volume of fresh transfection medium (RPMI) to acheive the required cell density (20 mio/ml).
4. Add 0.5 mL of the cell suspension to the tube with DNA and mix orbitally.
5. Add the PEI (30 ug) to the Cell+DNA mixture as soon as possible, flick 3 times.
6. Place in the incubator at 37°C.
7. Dilute the transfections with 9.5mL of growth medium (EX-CELL 293) 3 hours after the transfection.
HEK cells have been transfected with the pHY42 plasmid. The aim was to see if there was any expression difference with the CHO cells. We made two tubes that contained 50% of pGL-GFP and 50% of filler DNA to have a baseline fluorescence leakage. We also made two other ones that contained 50% melanopsin and 50% pGL-GFP.
Fluorescence measurement
Protocol: Fluorescence (Guava)
Prepare your samples by measuring their PCV (or estimating the cell amount according to the doubling rate). Dilute them with PBS in order to have between 200 and 500 cells/µl. Prepare at least one well that has seed cells.
Steps 1 to 4 are optional and should be done from time to time.
1. Trash the waste on the bottom right of the machine if it is full before you start.
2. Put tubes with bleach (detergent) at the right positions.
3. Run 'Cytosoft 5.3'
4. Click Clean and Shut Down -> This would take around 15 min.
5. After cleaning, click Guava Express Plus on the left column.
6. Go to Analysis mode and click 'Open Data Set'.
7. Go to the 'iGEM' folder and open the 'Setting' file.
8. Go to Acquisition - and hold here.
9. Go to the desktop and run WorkEdit 5.3.
10. Highlight the wells you are going to use, check "Acquire this sample".
11. Label them as Guava Express Plus, check the "Mix for 3 seconds", set the speed from high to medium. Optionally, fill the sample ID and the dilution factor.
12. Save and go back to Cytosoft 5.3.
13. Go to Acquisition, start the worklist.
14. Place the 96-well plate into the tray, make sure the A1 well is where it should be.
15. Name the file as 'Today's date_title'
16. When you are asked to adjust the settings, check a well that contains seed cells.
17. Compare with the worklist, check if the flow and the amount of cells detected are reasonable.
18. Click "Next step" and then "Resume".
19. Wait until all the wells are measured - data will be saved automatically.
20. Take the 96-well plate out and insert the tubes that are required for cleaning.
21. Go to Main menu and click 'Clean and shut down'.
The transfected cells were measured with the Guava flow cytometer 3 hours post-transfection and then transferred to a well plate under the Arduino. They were left under 10-second light pulses.
Western Blot result from 14.sep.12
Protocol: Western Blot
Gel Ingredients (choose percentage according to the size of the protein)
| 4-40 kDA | 20% |
| 12-45 kDA | 15% |
| 10-70 kDA | 12.5% |
| 15-100 kDA | 10% |
| 25-200 kDA | 8% |
| Separating gel | |
| Gel percentage | 7.5 % |
| 30% Polyacrylamide | 10 mL |
| 1.5M Tris (pH 8.8) | 10 mL |
| 10% Ammonium persulfate | 0.4 mL |
| 10% SDS | 0.4 mL |
| TEMED | 0.038 mL |
| H2O | 19.2 mL |
| Total volume | 40 mL |
| Stacking gel | |
| Gel percentage | 5 % |
| 30% Polyacrylamide | 1.36 mL |
| 1M Tris (pH 6.8) | 1 mL |
| 10% Ammonium persulfate | 0.08 mL |
| 10% SDS | 0.08 mL |
| TEMED | 0.008 mL |
| H2O | 5.44 mL |
| Total volume | 8 mL |
Preparing Protein Samples
1. Centrifuge around 5 million cells (of any volume) at 2,500 rpm for 10 min.
2. Discard the supernatant with a vacuum pump.
3. Resuspend the cell pellet with 1x PBS and centrifuge it at 2,500 rpm for 10 min.
4. Discard the supernatant with a vacuum pump.
5. Add appropriate amount of lysis buffer depending on the pellet size (for a 20 mg pellet, 150 µl of IP lysis buffer).
6. Keep the lysed sample on ice for 10 min - flick every 3 minutes.
7. Add 3x SDS lysis buffer (for a 20 mg pellet, 75 µl).
8. Incubate the sample for 5 minutes at 95 degrees, to denature proteins.
Preparing loading samples
1. Load the ladder (7 µl is the recommended volume).
2. Complete sample volume to 50 µl.
3. Load the samples.
I. SDS Gel electrophoresis
1. Prepare the separating and stacking gel solutions without APS and TEMED.
2. Add APS and TEMED to the separating gel solution only when the SDS kit is ready to be used, they are time-sensitive. Move the solution inside of the setup. Add some distilled water on top of it.
3. After 20-30 mins, remove the water and check whether the gel has solidified. Don't move to the next step until it does.
4. Add TEMED to the stacking gel solution, pour it on top of the solidified separating gel.
5. Insert a stack carefully and leave it for 20-30 mins.
6. Take the stack out and fill the kit with SDS loading buffer.
7. Load the samples.
8. Add more loading buffer, set the voltage to 80 Volts. Leave for 1.5 hours.
II. Membrane transfer
1. Prepare a membrane transfer kit.
2. Take the gel out of the SDS kit and put it on the membrane paper.
3. From bottom to top, assemble the components in the following order: 1) Sponge - 2) Blot paper - 3) Membrane - 4) Gel (Pour some M-transfer buffer on the gel) - 5) Blot paper again - 6) Sponge again.
4. Close the sandwich, set the voltage to 20 V. Leave for 30 mins - 1 hour.
5. Discard the gel. Leave the membrane in 5% skim milk with 30ml of TBST buffer (blocking buffer, to achieve the 5%, add 1.5 g of skim milk powder to the buffer) for one hour.
III. Antibody tagging
1. Discard the blocking buffer, leave only 5ml of it. Add primary antibody with a ratio of 1:1000 or 1:2000 (5 µl of antibody in 5 ml of buffer gives 1:1000)
2. Leave the mix overnight at 4 °C.
3. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).
4. Dilute the secondary antibody (for example, goat anti-rabbit antibody) to 1:2000 in 5% skim milk buffer. Add it. Leave at room temperature for 2 hours.
5. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).
6. Reveal the protein bands in the dark room.
- 48h post-transfection of LovTAP-VP16
- Lane 1: Ladder
- Lane 2: Empty
- Lane 3: VP16 only
- Lane 4: 45 µl untransfected CHO
- Lane 5: 45 µl LovTAP Sample 1 (100% LovTAP)
- Lane 6: 45 µl LovTAP Sample 2 (95% LovTAP + 5% GFP)
- Lane 7: 45 µl LovTAP Sample 2 (95% LovTAP + 5% GFP)
- Lane 8: 45 µl LovTAP Sample 3 (100% LovTAP)
- Lane 9: 45 µl LovTAP Sample 3 (100% LovTAP)
- Lane 10: 45 µl untransfected CHO
- 72h post-transfection of LovTAP-VP16
- Lane 1: Ladder
- Lane 2: Empty
- Lane 3: VP16 only
- Lane 4: Empty
- Lane 5: 35 µl untransfected CHO
- Lane 6: Empty
- Lane 7: 35 µl LovTAP Sample 1 (100% LovTAP)
- Lane 8: 35 µl LovTAP Sample 2 (95% LovTAP + 5% GFP)
- Lane 9: 35 µl LovTAP Sample 3 (100% LovTAP)
- Lane 10: 35 µl LovTAP Sample 3 (100% LovTAP)
The first membrane development should have shown a LovTAP-VP16 band in lanes 5-9 and not in lanes 4 and 10.
No such band has been observed.
Also, we could not find any special band in the second membrane, which should have shown a difference between lane 5 and lanes 7-10.
No Lovtap-VP16 expression has been confirmed in our transfected CHO cells.
