Objective: try to detect CNTN1 in neuroblastoma cells (indiferentiated) using a higher amount of protein. Try a precast gel 4-15%.
Samples/wells:
- Marker
- HTB11 cells (23.04.19): 22,5 ul + 7,5 ul laemmli 4x
- neuroblastoma neurons -HTB11 dif.- (27.03.19): 22,5 ul + 7,5 ul laemmli 4x
- swine sciatic nerve (26.03.19): 22,5 ul + 7,5 ul laemmli 4x
- mouse brain RIPA (11.04.19): 22,5 ul + 7,5 ul laemmli 4x
- Recombinant protein CNTN1: 8 ul + 14,5 ul RIPA + 7,5 ul laemmli 4x
Protocol:
- We have used a precast gel (4-15% achrylamide) with 10 wells (Bio-Rad)
- Warm the samples at 96ºC for 5 minutes
- Load samples in the wells (30ul per well)
- Run with running buffer 1x at 20 – 40mA
- Transfer to a PVDF membrane with Transblot Turbo (using the commercial buffer) –> mixed proteins protocol
- Blotting: wash the membrane with water.
- Blocking: blocking buffer Casein – TBS 1:1 1h
- Primary antibodies (diluted in Casein-TBS 1:1) –> overnight at 4ºC:
- Ac anti-CNTN1 (rabbit) 1/200
- Ac anti-Bactin (mouse) 1/20000
- Wash with TBS-tween0’1% (3×5′)
- Secondary antibodies GAR800 + GAM800 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:
Los geles precast de Bio-Rad son más delgados (0’75) que los que usamos habitualmente (1,5). Por eso las proteínas se transfieren mejor, y hemos podido detectar la CNTN1 en las células HTB11 (diferenciadas e indiferenciadas).
Por otro lado, no hemos podido detectar la CNTN1 en el lisado de nervio ciático de cerdo. Quizás el Ac anti-CNTN1 que hemos usado no es capaz de detectar la CNTN1 del cerdo.
