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Contents |
Passaging
Protocol: Cell Passaging
These are the general mammalian cell passaging rules, they work for CHO cells and HEK cells.
The cells should be left to grow for two days, after what their density should be reduced and the medium should be changed.
We usually bring them either to 2 mio/ml, either to 1 mio/ml.
- Put the volume of medium that corresponds to the amount of cells you'd like to passage at 37°C, leave it there to heat for 10 minutes (more for bigger volumes, less for smaller).
- Measure the PCV of your current cell sample. Usually, the cells double every day, so you can estimate their number, but a PCV is more precise. Take 200 µl of cell suspension in a mini-PCV tube, centrifuge them (1 min at 5000 rpm), the cells will fall down in the thin tube at the bottom. Use the VoluPAC Reader to measure the PCV (the volume in µl the centrifuged cells take).
- If you're dealing with HEK cells, the PCV is also the number of millions of cells/ml you have.
- If you have CHO cells, divide the PCV by 0.4 to obtain the mio/ml cell density.
- Take the volume that will contain the amount of cells you need from the original sample in a centrifuge tube, centrifuge it at 1500 rpm for 3 minutes. A pellet will form.
- Remove the supernatant (under the hood) either with the vacuum pump, either by simply discarding it.
- Resuspend in 10 ml of warmed medium (pipet up and down)
- Add the remaining medium, mix again
- Split the cultures into 10 ml samples in separate tubes, put them in the shaker at 37°C, passage again after 2 days.
We passaged the CHO cells, and made a HEK culture from the lab stock.
Western Blot result from 8.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.
- Lane 1: Ladder
- Lane 2: 10 (or 30) LovTAP Sample 1 (100% LovTAP)
- Lane 3: 10 (or 30) LovTAP Sample 2 (100% LovTAP)
- Lane 4: 10 (or 30) LovTAP Sample 3 (50% LovTAP + 50% filler)
- Lane 5: 10 (or 30) LovTAP Sample 4 (50% LovTAP + 50% filler)
- Lane 6: 20 (or 40) LovTAP Sample 1 (100% LovTAP)
- Lane 7: 20 (or 40) LovTAP Sample 2 (100% LovTAP)
- Lane 8: 20 (or 40) LovTAP Sample 3 (50% LovTAP + 50% filler)
- Lane 9: 20 (or 40) LovTAP Sample 4 (50% LovTAP + 50% filler)
- Lane 10: 20 (or 40) non-transfected CHO cell as a control + 5 µg of VP16 only for the first membrane
The sample was taken 24 hours post-transfection.
The membrane was dirty because of a wrong secondary antibody and did not wash well.
Cannot confirm the LovTAP-VP16 expression.
This result is not conclusive because of the membrane damage.
