Objective: To detect serum CNTN1 in a CIDP patient. Sera CNTN1 is detected by ELISA but we do not have any confirmatory technique. We have tried to perform WB to detect it in sera, being unsuccessful.
Sample: 17-183 (200pg/mL of sera CNTN1 detected by ELISA in Amsterdam – Dr. Luuk Wieske)
PROCEDURE:
Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo Scientific num. 88805) – following the kit instructions.
- Pre-wash beads two times with 1x Modified Coupling buffer
- Bind antibody to beads for 15 min
- Wash beads for 15 min
- Crosslink antibody to beads with DSS for 30 minutes
- Wash beads 3 times with elution buffer followed by 2 washes with IP Lysis/Wash Buffer
- Incubate sera with antibody-crosslinked beads for 1-2h at RT or ON 4ºC
- Wash beads 2 times with IP Lysis/Wash Buffer and 1 time with ultrapure water
- Elute bound antigen (Lane Market Sample Buffer)
We will perform the WB next week.
