Objective: To detect protein CNTN1 in blood samples. The first experiment was not successful and we are going to try changing some conditions (condition 1) and chinging the order of the antibodies (condition 2).
Materials:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human CNTN1 protein
- AF904 abcam anti-CNTN1
- AB66265 Abcam anti-CNTN1
- DAG-peroxidasa Ref. 705-035-147 (box 27)
- GAR-peroxidasa P0448
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4 0.25%
- PBS-Tween20 0.1%
PROCEDURE:
- Coat ELISA plate with Ab66265 for Condition 1 and AF904 for condition 2 (1ugr/mL) in sodium carbonate buffer (pH 9.6) and incubate at 4ºC rocking.
- The day after, wash plate 3xPBS-Tween 0.1% and block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (200uL/well) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add AF904 (1ugr/ml) in milk 5% and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add DAG-peroxidase diluted 1/2000 in milk 5%
- Wash 3xPBS-Tween
- TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
- CONDITION 2:
- The day after, wash plate 3xPBS-Tween 0.1% and block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (200uL/well) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add Ab66265 (1ugr/ml) in milk 5% and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add GAR-peroxidase (P0448) diluted 1/2000 in milk 5%
- Wash 3xPBS-Tween
- TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS:
The ELISA has not worked correctly.
