Objective: to do an immunoabsorption of NF155 in a serum from a patient with antibodies anti-NF155.
22.11.2018: Coating HEK293
- Prepare 4 6-well plates
- Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS
- Trypsin-EDTA a plate of HEK293 –> For every well: 400.000 cells
- Incubate at 37ºC for 24h.
23.11.2018: Transfection
Transfect two 6-well plate with NF155, and one with NF186 (the other one is not transfected)
- [NF186] = 0,983 µg/µL. 4 µg /well –> 25 µL + 750 µL Optimem
- [NF155] = 1,104 µg/µL. 4 µg /well –> 43’5 µL + 1,5 mL Optimem
- Lipofectamine: 6 µL/well. 108 µL + 2,25 ml Optimem
- Incubate the DNA and the lipofectamine at RT for 5 min.
- Unify every DNA with the appropriate amount of Lipo and incubate at RT for 30 min.
- For every well: 2 ml of HEK medium and 250 µL of the mix of DNA and Lipo.
- Incubate O/N at 37ºC.
24.11.2018: Fixing and freezing
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Freeze at -80ºC (without liquid)
26.11.2018: Immunoabsorption
Sample: serum from J.C. de Haro Mateo (18-858 ICQ)
- Dilute the serum 1/50 in PBS
- Put 0’5 ml in the first well of every plate
- Incubate 1 hour in every well (at RT) –> in the case of NF155, incubate 30 mins in every well (12 wells)
- Freeze the immunoabsorpted samples at -20ºC
