Objective: to do an immunoabsorption of NF155 (and NF140), in a serum from a patient with antibodies anti-NF155.
29.10.2018: Coating HEK293
- Prepare 2 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.
30.10.2018: Transfection
Transfect one 6-well plate with NF155, and the other one with NF140.
- [NF140] = 1,034 µg/µL. 4 µg /well –> 23,2 µL + 750 µL Optimem
- [NF155] = 0,5 µg/µL. 4 µg /well –> 48 µL + 750 µL Optimem
- Lipofectamine: 6 µL/well. 72 µL + 1’5 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.
31.10.2018: Immunoabsorption
Sample: IgGs from J.C. de Haro Mateo (18-851 ICQ)
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Dilute the IgGs 1/100 in PBS
- Put 0’5 ml in the first well of every plate.
- Incubate 45 minutes in every well (at RT)
- Freeze the immunoabsorpted samples at -20ºC
