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Contents |
Fluorescence measurements of melanopsin
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'.
- Measurement times:
- 09:00 Take T = 14h sample of first well plate transfer
- 16:00 Put tube spin cells in well plate (24h after transfection).
- 19:00 Take T = 24h sample of first well plate transfer. Take T = 3h sample of second well plate transfer
Western Blot on LovTAP
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.
A new Western Blot was started with the LovTAP-transfected cells.
