Objective: We use the product of the IP done on 28.03.2019 (C+, C-), to see if we can detect the CNTN1 in the positive control. Moreover, we try to detect CNTN1 in neuroblastoma neurons (HTB11 differentiated).
Samples/wells:
- Marker
- IP C+: 35 ul
- Flow through IP C+: 32 ul + 8 ul Lane Sample Marker 5x (LSM)
- IP C-: 35 ul
- Flow through IP C-: 32 ul + 8 ul Lane Sample Marker 5x
- Swine ciatic nerve lysate (total): 50ug –> 10 ul + 8 ul LSM5x + 22 ul lyssis buffer (kit IP)
- Recombinant protein CNTN1: 20 ul + 8 ul LSM5x + 12ul
- Marker
- Neurobl. neurons: 50 ug –> 11 ul + 4 ul RIPA + 15 ul Laemli2x
- Recombinant protein CNTN1: 20 ul + 20 ul Laemli2x
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 8-10): Ac anti-CNTN1 (Rabbit) 1/500
- Wash with TBS-tween0’1% (3×5′)
- Secondary antibody 1/7500 (diluted in Casein-TBS tween0’1%): 1h at RT:
- Mb1 (wells 1-7): DAG800
- Mb2 (wells 8-10): GAR800
- Wash with TBS-tween0’1% 1x (2×5′)
- Wash with TBS 1x (1×5′)
- Read with Odyssey equipment
Result:
We only can see a band in the recombinant protein wells (that correspond to CNTN1).
Repeat using B-actin as control.
