20180726: HEK293 IP (with Pierce Classic Magnetic IP Kit)

25.07.2018: NF155 Transfection

• The day before, the cells were seeded at 3 milion cells/plate (100 mm of diameter)  
• For every plate of 100 mm of diameter –> 13.4 microg of DNA + 20 microL of Lipofectamine.
  
  • In this case (for 2 plates)
    • 31.3 microL of DNA in 834 microL of Optimem.
    • 40 microL of Lipo in 834 microL of Optimem.

26.07.2018: IP

• Incubate the cells 1h at 37ºC (in the incubator) with serum diluted 1/100 in culture medium. 
  • Positive control (LAC): neurofascin 155 positive 
  • Negative control
• Carefully remove culture medium from confluent cells.
• Wash the cells once with PBS.
• Add 0.8 ml of ice-cold IP Lysis/Wash Buffer to the cells. Incubate on ice for 5 minutes with periodic mixing
• Transfer the lysate to a microcentrifuge tube and centrifuge at ~13,000 × g for 10 minutes to pellet the cell debris.
• Transfer supernatant to a new tube.
• 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 IP Lysis/Wash Buffer 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 antigen sample/antibody mixture 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 and save for analysis.
• Add 500μL of IP Lysis/Wash Buffer 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 100μL 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)

27.07.2018: Electrophoresis

• Make the SDS gels:
  • Stacking gel (concentración): 4 %
  • Separating gel (resolución): 10 %
• Heat the samples at 96-100ºC for 5 minutes
• Put 30 microl of each sample in each well, and run at 20 mA
• 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)
  

RESULTS: there are no differences between the positive and the negative controls. There are a lot of protein in the top of the gel, so maybe it has not entered well. 

Repeat the experiment but with CNTN1 (instead of NF155), and do the IP with Agarose and with Magnetic beads at the same time (to confirm that this new method works better than agarose method).