2022.07.27 IgG IP CIDP 98 sera in DRG neurons

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. add 50ul of elution buffer to the tube. incubate the tube at RT with mixing for 10 minutes.

10. magnetically separate the beads and save the sueprnatant containing the target antigen.

11. to neutralize the low pH , add 5ul of neutralization buffer for each 50ul of eluate

Sample: 21-2-1567 (CIDP 98) –> Patient with CISP

Excel reusultados

Print Friendly, PDF & Email

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.