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. In the last experiment on 04-05/02/2020 we’ve seen a possible band for CNTN1, but the sample has a strong background and we are going to remove IgG and albumin first.
SAMPLES: 17-183 (by ELISA – 2141 pg/mL – high), recombinant protein CNTN1.
Preparation of the sample: Protein precipitate from 2020/02/04 (performed with acetone) and we are going to remove IgG and albumin with ProteoPrep Blue Albumin and IgG depletion Kit (Sigma-Aldrich) – Product Number : PROTBA, following the isntructions from the kit. Protein cuantification 3.25ug/uL.
PROCEDURE for Western Blot (20200211-12):
- 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); marker, 50ug of protein sample, 75ug of protein sample and recombinant sCNTN1 protein.
- 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 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 RAG800 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 have a possible band for sCNTN1, we will try to perform it with different CIDP samples that don’t have detectable sCNTN1 by ELISA and see if it is not a unspecific band.
