Objectives: to test if the WB with non-denaturing conditions is better to detect de NF155 antibodies in serum.
Samples:
- Protein extract of HEK293 cells transfected with NF155 (20180627: Extraction and quantification of protein (HEK293 NF155 extract)
- Protein extract of non-transfected HEK293 (20180627: Extraction and quantification of protein (HEK293 NF155 extract)
Protocol:
- Prepare the Native-page gels
- 8 % resolution gel
- Tris pH 8.8 3M: 1.25ml
- Acrilamide 40%: 2 ml
- H2O: 6.65ml
- PSA 10%: 100ul
- Temed: 10ul
- 4 % concentration gel
- Tris pH 6.8 2M: 0.75ml
- Acrilamide 40%: 1 ml
- H2O: 8.25 ml
- PSA 10%: 100ul
- Temed: 10ul
- 8 % resolution gel
- Prepare the samples (dilute the samples in Native Sample Buffer from Bio-Rad):
| Mostra (µl) | Pierce IP Lysis Buffer (µl) – fins 15 µl | Native Sample Buffer 2x (µl) | |
| Prot. Recombinant NF155 1 µg | 9,4 | 5,6 | 15 |
| HEK NF155 30 µg | 3,2 | 11,8 | 15
|
| HEK NF155 50 µg | 5,4 | 9,6 | 15
|
| HEK Sense transf 30 µg | 2,7 | 12,3 | 15
|
- Load samples in the wells (30ul per well) –> the 4 samples are loaded 2 times (one for use with control positive serum and one for use with control negative serum)
- Run with Native Page Buffer 1x (from Bio-Rad) at 20 – 40mA
- Transfer to a PVDF membrane with Transblot Turbo (using the commercial buffer) –> high proteins protocol
- Blotting: wash the membrane with water and cut it
- Blocking: blocking buffer Odyssey – PBS 1:1 1h
- Serum 1/100 (Odyssey-PBS) 1h
- Positive control (15-195)
- Negative control
- Wash with PBS 1x (3×5′)
- Primary antibody: anti-NF155 1/1000 (1 h)
- Wash with PBS 1x (3×5′)
- Secondary antibodies (GAH800 + DAC680) 1/7500 1h
- Wash with PBS 1x (3×5′)
- Read with Odyssey equipment
RESULTS:
- Transfer: han quedat proteïnes altes al gel –> s’ha de transferir a més potèn cia
- Anti-NF155 antibody (vermell): es veu una banda als dos carrils de proteïna pura, però es veu poca als carrils de cèls HEKs transfectades.
- Suero (verd): el control positiu marca molt NF al pou amb proteïna pura. Als altres pous no es veu tanta diferència
- Propera vegada: transferir a més potència, deixar córrer més, carregar tota la proteïna que es pugui
Imatges:
- Gel 1.1: suero Control negatiu
- Gel 1.2: suero control positiu
- Gel 2.1: anti-NF control negatiu
- Gel 2.2: anti-NF control positiu
