OBJECTIVE: To detect soluble protein CNTN1 in pacients’ sera (determined by commercial ELISA in samples from Amsterdam, by Dr. Wieske)
SAMPLES:
- 17-183 (by ELISA – 2141 pg/mL – high)
- 17-177 (by ELISA – 1026pg/mL -normal)
- 17-182 (559pg/mL – low)
- CIDP 67 (not detectable by ELISA)
To prepare samples:
- First approach: Mix 22.5uL of sera + 7.5 Laemmli. When we warm it at 96ºC the mixture solidifies. We cannot perform it beacuse first, we have to precipitate proteins!
- Second approach: Dilute the sera 1/10 (2.5uL of sera + 20.5uL RIPA + 7.5uL Laemmli). We perform the experiment with this second approach.
PROCEDURE:
- 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 – TBS 1:1 1h
- Primary antibody AF904 (diluted in Casein-TBS 1:1) –> 1h at RT or overnight at 4ºC
- Wash with TBS-tween0’1% (3×5′)
- Secondary antibody DAG680 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 did not detect protein in any of the samples. CNTN1 sera concentration is very low (maximum 2000pg/mL) and we dilute the sera to perform the WB.
We are going to precipitate protein from sera and then perform the WB. If we can not detect it, then we are going to try to perform it depleting Albumin and IgG and/or biotynilating the antibody.
