Samples:
- 15-198 (CNTN1+)
- Negative control
Nerve lysate:
- Clean and disgregate the tissue as much as possible
- Grind (crush) the nerves with a Potter Homogenizer (in 1 ml of Lysis Buffer from the Pierce Magnetic IP kit + protease inhibitors).
- We use 400 microl of the lysate for every sample, and we keep the rest at -20ºC.
Pierce Magnetic IP Kit
- Incubate the tissue with the serum of the patient to be analyzed 1h at RT in constant rotation.
- For IgG: 1/100
- To prepare the magnetic beads: Put 25 μl of Pierce Protein A / G Magnetic Beads into 2 eppendorfs
- Add 175 μl of IP Lysis / Wash Buffer and vortex (gently)
- Put the tube in the magnetic support. Remove the supernatant (without removing the eppendorf from the support)
- Add 1 ml of IP Lysis / Wash Buffer and vortex (gently). Put back in the magnetic support and remove the supernatant.
- Add the antigen-antibody mixture (first part) to the eppendorf with the washed magnetic beads and incubate at room temperature for 1 hour in agitation (rotation).
- Put the eppendorfs on the magnetic support and remove the supernatant.
- Add 500 μl of IP Lysis / Wash buffer (with protease inhibitors) and mix. Put the eppendorfs on the magnetic support and remove the supernatant.
- Repeat washing
- Add 500 μl of ultra pure distilled water and mix. Put the samples on the magnetic support and remove the supernatant.
- Add 100 μl of Lane Marker Sample Buffer (diluted 5x in distilled water) to the tube and heat to 96 – 100 ° C for 10 minutes
- The amount of Lane Marker Sample Buffer can be changed depending on the objective of the IP.
- Put the eppendorfs in the magnetic support (the magnetic beads will separate from the antigen-antibody solution)
- With the supernatant perform the acrylamide gel electrophoresis (save the samples at -20ºC until the electrophoresis is done)
