Samples:
- 17-219 (2000pg/mL of sera CNTN1 detected by ELISA in Amsterdam – Dr. Luuk Wieske)
- plasma from a CNTN1+ CIDP patient (no detectable CNTN1 in sera)
- plasma from GM1+ GBS patient (ELISA not performed).
PROCEDURE Co-IP:
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)
The differences performed in this experiment regarding the previous Co-IP are:
- We do not use IP Lysis/Wash Buffer to dilute sera in step 6.
- We use more uL of beads (50uL for each IP reaction instead of 25uL), and for this reason we use x2 uL of all reactions ad washes, except for primary antibody: 25uL (5ug) for each IP reaction.
We froze the IP and perform the WB on 2020/06/05. We perform two gels:
- One for staining with NOVEX (with sample 17-219 and CNTN1 protein) to send to mass spectometry and corroborate that we have CNTN1 protein.
- One to perform WB) with the 3 samples (17-219, plasma from GBS and plasma from CNTN1+ CIDP). This gel for WB broke into a lot of pieces and we cannot perform this part of the experiment.
For Staining the sample with NOVEX:
- Wash with H20d x2
- Fix 15 min in slow agitation (Fixation solution: 10mL metanol, 2mL acetic aceid, 8mL H20d)
- Wash wish H20 x2
- Stain with NOVEX (Colloidal Blue Staining Kit Invitrogen LC6025) dor 3-12h in slow agitation (NOVEX solution: 4mL Metanol, 4mL Satiner B, 4mL Stainer A, 11mL H20d) for 3-12h.
- Fade with H20d in slow agitación ON 4ºC
- Cut the bands and send them to mass spectometry (A and B possible CNTN1 and C recombinant protein CNTN1)
RESULTS: We found protein CNTN1 in band A. Find the proteomic results here.
