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: 15 ul + 15 ul T.Nicholson
- Swine ciatic nerve lysate: 15 ul + 15 ul laemli 2x
- Swine ciatic nerve lysate: 15 ul + 9 ul lysis buffer IP + 6 ul Lane Sample marker 5x
- Swine ciatic nerve lysate (resuspended in Nicholson): 30 ul
- Neuroblastoma neurons: 15 ul + 15 ul T.Nicholson
- Neuroblastoma neurons: 15 ul + 15 ul laemli 2x
- Neuroblastoma neurons: 15 ul + 9 ul lysis buffer IP + 6 ul Lane Sample marker 5x
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 Ac anti-CNTN1 (rabbit) 1/1000 + Ac anti-Bactin 1/20000 (diluted in Casein-TBS 1:1) –> 1h at RT or overnight at 4ºC:
- Wash with TBS-tween0’1% (3×5′)
- Secondary antibodies GAR800 + GAM680 1/7500 (diluted in Casein-TBS tween0’1%): 1h at RT
- Wash with TBS-tween0’1% 1x (2×5′)
- Wash with TBS 1x (1×5′)
- Read with Odyssey equipment
RESULT:
We can only see the band that corresponds to CNTN1 in the well with recombinant protein.
In all the other wells there is B-actin, suggesting that the lysates and the cell suspensions are well done. Maybe we have to put a higher amount of protein to detect the CNTN1.
