OBJECTIVE: To detect soluble protein CNTN1 in pacients’ sera (determined by commercial ELISA in samples from Amsterdam, by Dr. Wieske). We are going to try to detect CNTN1 in one sample that have the highest concentration.
SAMPLES: 17-183 (by ELISA – 2141 pg/mL – high), recombinant protein CNTN1.
PROCEDURE:
To prepare the sample: PROTEIN PRECIPITATION: 2020/02/04
- 900uL of acetone at -20ºC for each 100uL of sera
- Vortex
- Store 1h at -20ºC
- Spin for 10 min at 13.000g (4ºC)
- Eliminate the supernatant
- Mix the pellet with 500uL of acetone at -20ºC
- Spin for 10 min at 13.000g
- Aspirate and left the eppendorf opened for 30min to evaporate acetone.
- Mix with RIPA and proteasa inhibitors
- Incubate 30 min in ice + vortex
- Spin for 10 min at 13.000g
- The supernatant is what we needed, if something precipitate we have to remove it.
- Quantify the protein (595nm): 35.34mg/ml
PROCEDURE for Western Blot (20200205):
- Use precast gels from BioRad (10 wells, 4-15 %)
- Warm the samples at 96ºC for 5 minutes
- Load samples in the wells (30 ul per well)
- Run with commercial running buffer 1x at 20 – 40mA
- Transfer to a nitrocelullose membrane with Transblot Turbo (using the commercial buffer) –mixed proteins protocol
- Blotting: wash the membrane with water and cut it
- Blocking: blocking buffer Casein – PBS 1:1 1h
- Primary antibody AF904 (diluted in Casein-PBS 1:1) –> 1h at RT or overnight at 4ºC
- Wash with PBS-tween0’1% (3×5′)
- Secondary antibody DAG680 1/7500 (diluted in Casein-PBS tween0’1%): 1h at RT
- Wash with PBS-tween0’1% 1x (2×5′)
- Wash with PBS 1x (1×5′)
- Read with Odyssey equipment
RESULTS: We’ve seen possible band but it seems to be unespecific. We are going to perform stripping and repeat it with another primary antibody CNTN1.
