Protocol using Pierce Classic Magnetic IP Kit
1.incubate serum 1h at 37ªC, in 5 plates con DRG neurons. Serum dilution 1/100. 20ul of serum in 2ml of medium.
2. discarge medium and add 800ul of IP lysis/Wash buffer with prot inh 1x and shake hard during 15min in the cold room
3.transfer the lisate to 2-3 2ml eppendors using a scraper and centrifuge 10min at 13000g
4.during the previous step wash Pierce Prteoin A/G Magnetic beads: add 25ul of magnetic beads in a 2ml epp, add 175ul of IP buffer (without PI) and vortex to mixt. place the tube into a magnetic stad to collect the beads. remove and sicard the supernatant. add 1mL of IP buffer to the tube. Invert the tube several times or gently vortex to mix for 1 minute. Colletct beads with magnetic stand. Remove and discard the supernatant.
5. Add the supernant from the lisate to the washed Magnetic beads and incubate 1h at RT in rotation
6.collect the beads with a magnetic stadn, remove the unbound sample
7.add 500ul of IP buffer to the tube and gently mix. collect the beads on a magnetic stand and discard the supernatant. repete this wash twice. In this step mix all 2-3 eppendorfs in one
8.add500ul of ultrapure water to the tube and gently mix. collect the beads on a magnetic stand and discard the supernatant.
9. a. add elution buffer to the tube. incubate the tube at RT with mixing for 10 minutes.
magnetically separate the beads and save the supernatant containing the target antigen.
to neutralize the low pH , add neutralization buffer. –> posteriormente la muestra fue desnaturalizada con bmercapto y corrida en gel con laemli –> vemos banda en 120 kDa aprox
9. b. lane marker 50ul and heat the samples at 96-100º in a heating block for 10 minutes. Magnetically separete the beads and save the supernatant-containing target antigen –> producto corrido en gel. Vemos muestra no desnaturalizada y banda en 110 kDa aprox correspondiente a CNTN1
Sample: 21-2-1495 (CNTN1+)
Bandas del gel enviadas al masas como se detalla en la figura
EXCEL RESULTADOS IP
