20200626: CNTN1 IP (with Pierce Classic Magnetic IP Kit)
Place 25μL (0.25mg) of Pierce Protein A/G Magnetic Beads into a 1.5mL microcentrifuge tube.
Add 175μL of IP Lysis/Wash Buffer to the beads and gently vortex to mix.
Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
Add 1mL of PBS to the tube. Invert the tube several times or gently vortex to mix for 1 minute. Collect beads with magnetic stand. Remove and discard the supernatant.
Add the monoclonal antibody (1- CNTN1 AF904 0.2 ug/ul – 25ul=5ug) (control 2- LRP4 0.1 ug/ul – 25ul=2.5ug) diluted in PBS to a 100ul volume (with proteasa inhibitors 1x) to the tube containing pre-washed magnetic beads and incubate at room temperature for 1 hour with mixing.
Collect the beads with a magnetic stand, remove the unbound sample.
Add 100μL of PBS to the tube and gently mix. Collect the beads and discard the supernatant. Repeat this wash with 300ul of PBS.
Add de rhCaspr1 protein (6ug/mL) (1- 1000 ul) (2- 500ul) to the tube containing magnetic beads + Ab (1-) (2-) and incubate at room temperature for 1 hour with mixing.
Collect the beads with a magnetic stand, remove the unbound rhCaspr1 protein –> store at 80º until the ELISA
Add Add 500μL of IP Lysis/Wash Buffer (with proteasa inhibitors 1x) to the tube and gently mix. Collect the beads and discard the supernatant. Repeat this wash twice.
Add 500μL of ultra pure water to the tube and gently mix. Collect the beads on a magnetic stand and discard the supernatant.
Add 60μL of Lane Marker Sample Buffer (diluted five-fold with purified water) to the tube and heat the samples at 96-100ºC in a heating block for 10 minutes. Magnetically separate the beads and save the supernatant-containing target antigen.
The samples are frozen at -20ºC (until the electrophoresis)
