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. Based on experiment from WB 020200205. Using the nitrocellulose from 20200204-05 experiment, we will perform stripping.
SAMPLES: 17-183 (by ELISA – 2141 pg/mL – high), recombinant protein CNTN1.
PROCEDURE:
- Stripping buffer diluted 1/5 in destilated water for 10 minutes (NewBlot Nitro Stripping buffer (Odyssey, nº 928-40030) )
- Wash with PBS-tween0’1% (3×5′)
- Blocking: blocking buffer Casein – PBS 1:1 1h
- Primary antibody rabbit (diluted 1:200 in Casein-PBS 1:1) –> 1h at RT or overnight at 4ºC
- Wash with PBS-tween0’1% (3×5′)
- Secondary antibody GAR800 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 for sCNTN1, but the sample has a strong background. We are going to remove IgG and albumin first and repeat the experiment.
