| Step 1 |
Parameter |
Amount/Well (µL) |
Concentration |
Buffer* |
Incubation |
| 1 |
Coat wells
STNFR1 |
50 |
0.25 µg/mL |
A |
16 hours
25°C |
| 2 |
Wash
TWEEN-20 |
140 |
1% |
B |
10 minutes
3 times,
25°C |
| 3 |
Block wells BSA |
140 |
10% |
B |
1 hour,
37°C |
| 4 |
Wash
TWEEN-20 |
140 |
1% |
B |
10 minutes
3 times,
25°C |
| 5a |
Saturation TNFα |
50 |
Serial dilutions from 100nM |
C |
2 hours,
25°C |
| 5b |
Inhibition |
|
Serial dilutions
from 100 nM |
C |
2 hours,
25°C |
| TNFa |
25 |
14 nM (7 nM
final) |
D |
2 hours |
| inhibitor |
25 |
Serial Dilution |
D |
25°C |
| 6 |
Wash
TWEEN-20 |
140 |
1% |
B |
10 minutes
3 times,
25°C |
| 7 |
Anti-
TNFα(biotin) |
50 |
0.25 mM |
C |
1 hour,
25°C |
| 8 |
Wash
TWEEN-20 |
140 |
1% |
B |
10 minutes
3 times,
25°C |
| 9 |
TMB |
40 |
|
|
30 minutes,
25°C |
| 10 |
Sulfuric acid |
40 |
0.16 M |
|
20 minutes,
25°C |
| 11 |
Read at 450 nm minus 550 nm |
*Buffer A: 50 mM Carbonate-bicarbonate (pH 9.6), B: PBS, C: 4% (v/v) BSA in
PBS (pH 7.3), D: 4% BSA (v/v) in PBS, 2.5% (v/v) DMSO (pH 7.3). |
