20200221: Western Blot CNTN1 in sera

• OBJECTIVE: To detect soluble protein CNTN1 in pacients’ sera (determined by commercial ELISA in samples from Amsterdam, by Dr. Wieske). Based in a possible CNTN1 band detected on the experiment on 20200211, we are going to repeat it with different CIDP patients to see if we can detect a differential band.

SAMPLES:

• 17-183 (CNTN1 by ELISA – 2141pg/mL – high),
• 17-224 (CNTN1 by ELISA – 1151pg/mL- normal)
• 17-182 (CNTN1 by ELISA 559pg/mL – low)
• CIDP 67 (no detectable CNTN1 in sera)
• Recombinant protein CNTN1 (control)
  

Preparation of the samples:

1. Protein precipitatation with acetone (20200219), following the protocol and quantification of proteins (Cytoskeleton Kit):

• 17-183: 35.36ug/uL
• 17-224: 34.21 ug/uL
• 17-182: 33.75ug/uL
• CIDP 67: 30.15ug/uL

2. Remove IgG and albumin with ProteoPrep Blue Albumin and IgG depletion Kit (Sigma-Aldrich) – Product Number : PROTBA, following the instructions from the kit (20200220). Quantification of proteins (Cytoskeleton Kit):

• 17-183: 3.28ug/uL
• 17-224: 1.4625ug/uL
• 17-182: 3.825ug/uL
• CIDP 67: 1.275ug/uL

3. Centrifugal filter Unit (20200221):

To obtain a more concentrated sample (we need 75ug in the WB and maximum of sample is 50uL), we used the Centrifugal Filter Unit (30K) in samples 67 and 17-224:

• 17-224: 3.825ug/uL
• CIDP 67: 2.381ug/uL

PROCEDURE for Western Blot (20200221):

• Use precast gels from BioRad (10 wells, 4-15 %)
• Warm the samples at 96ºC for 5 minutes
• Load samples in the wells (50 ul per well); marker, 75ug of protein samples 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 cannot find any specific band for sCNTN1. We are going to perform an IP to try to detect CNTN1 in serum.