Objective: try to detect CNTN1 in neuroblastoma neurons (HTB11 differentiated), and in swine sciatic nerve lysate.
Samples/wells:
- Marker
- Recombinant protein CNTN1: 15 ul + 15 ul laemli 2x
- Swine ciatic nerve lysate (resuspended in Nicholson): 30 ul
- Swine ciatic nerve lysate: 15 ul + 15 ul laemli 2x
- Neuroblastoma neurons (lysate done in lysis buffer IP + LSM): 30 ul
- Neuroblastoma neurons (lysate done in Nicholson): 30 ul
- Neuroblastoma neurons (lysate done in RIPA+laemli 2x): 30 ul
- Marker
- Neuroblastoma neurons (lysate done in Nicholson): 30 ul
- Neuroblastoma neurons (lysate done in Nicholson): 30 ul
Protocol:
- Prepare the acrylamide gels:
| 8 % separating gel | 4 % stacking gel |
| Tris pH 8.8 3M: 1.25ml Acrylamide 40%: 2 ml H2O: 6.65ml SDS 20%: 50ul PSA 10%: 100ul Temed: 10ul | Tris pH 6.8 2M: 0.75ml Acrylamide 40%: 1 ml H2O: 8.25 ml SDS 20%: 50ul PSA 10%: 100ul Temed: 10ul |
- Warm the samples at 96ºC for 5 minutes
- Load samples in the wells (30-40ul per well)
- Run with running buffer 1x at 20 – 40mA
- Transfer to a PVDF membrane with Transblot Turbo (using the commercial buffer) –> high proteins protocol (changing the time to 13 min)
- Blotting: wash the membrane with water and cut it in 2 parts.
- Blocking: blocking buffer Casein – TBS 1:1 1h
- Primary antibodies (diluted in Casein-TBS 1:1) –> 1h at RT or overnight at 4ºC:
- Mb1 (wells 1-7): Ac anti CNTN1 (Goat) 1/500
- Mb2 (wells 8-9): serum positive control (15-198) 1/100
- Mb3 (well 10): serum negative control 1/100
- Wash with TBS-tween0’1% (3×5′)
- Secondary antibodies 1/7500 (diluted in Casein-TBS tween0’1%): 1h at RT
- Mb1 (wells 1-7): DAG800
- Mb2 (wells 8-9): GAH800
- Mb3 (well 10): GAH800
- Wash with TBS-tween0’1% 1x (2×5′)
- Wash with TBS 1x (1×5′)
- Read with Odyssey equipment
RESULT:
