PROTOCOL: Immunoprecipitation (Magnetic beads) + gel electrophoresis and staining

Pierce Magnetic IP Kit

• Incubate the culture or the tissue with the serum of the patient to be analyzed (for IgG 1/100):
  • If the IP is done in cultured cells:
    • incubate 1h at 37ºC (in the incubator). The dilution is done in the culture medium itself (on the plate)
    • Wash 1 time with PBS1x
    • Add 800 μl of IP Lysis / Wash buffer + protease inhibitors to each plate. Leave them in strong agitation during 5 min at 4ºC.
    • Transfer the lysate to a 2 ml eppendorf (in each eppendorf we will have the lysate of two plates) and centrifuge 10 minutes at 13000 g
  • If the IP is done in tissue: incubate fot 1h at RT in constant rotation. The dilution is done directly in the tissue suspension –> use aprox 1000 microg of protein for each sample (the proteins of the tissue were extracted with lysis buffer and protease inhibitors).
• To prepare the magnetic beads: Put 25 μl of Pierce Protein A / G Magnetic Beads in the number of eppendorfs that we will use.
• 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)

Electrophoresis

• Prepare the acrylamide gels:

• Heat the samples at 96ºC for 5 minutes
• Load samples in the wells (30-40ul per well)
• Run with running buffer 1x at 20 – 40mA
• Wash 3 times with PBS1x
• Fix for 15 min with:
  • 50 % methanol (10 ml)
  • 10 % acetic acid (2 ml)
  • 40 % dH₂0 (8 ml)
• Wash 3 times with PBS1x
• Stain the gel with NOVEX from 3 to 12 hours (Colloidal Blue Staining Invitrogen):
  • 4 ml Methanol
  • 4 ml Stainer A
  • 1 ml Stainer B
  • 11 ml  dH₂0 
• Discolour by washing with dH₂0 (overnight at 4ºC)