Protein A Beads and IgG Binding and Filtering (with SepCon Format)

Based on the SIGMA Product Information for “Protein A-Immobilized” (adapted for SepCon from Column Method)

The necessary components:

1) Buffer A:

  • 0.2M NaH2PO4 2.4g
  • 0.15M NaCl            8.8g
  • adjust to pH 8.0

2) Buffer B:

  • 0.2M Na2HPO4 25.7ml
  • 0.1M Citric Acid     24.3ml
  • DI H2O                    50.0ml
  • adjust to pH 4 (for human IgG)
  • adjust to pH 3 (we found that a pH of 4 doesn’t unbind IgG from beads; needs a lower pH)

3) Human IgG: reconstituted into 1mg/ml aliquots

4) Swollen Beads:

  • literature: “One gram of powder [protein A beads] typically swells to 3-4ml of hydrated gel”
  • In order to swell, though, one needs an excess of buffer A
  • Add ~11ml of buffer A
  • Let swell for at least 30 minutes at room temperature (we did ~4 hours)

5) A Transwell, to fit into the 24-well plate

Storage Note: The protein A beads must be stored in 20% ethanol (diluted from 100% concentration with buffer A)

Experiment in SepCon Format:

1) Rinse ethanol off of protein A beads (use ~20 CV)

  • Spin down protein A beads (2 minutes at 1500 rpm)
  • Aspirate.
  • Add 7ml buffer A
  • Shake
  • Spin down (2 minutes at 1500 rpm)
  • Aspirate.

2) Add IgG

  • Transfer beads to 2ml centrifuge tube
  • Add human IgG
  • Mix
  • Let sit in the refrigerator overnight, to allow plenty of binding time

3) Remove unbound IgG, until TECAN registers a neutral absorbance reading (~10CV)

  • Spin down (2 minutes at 1500 rpm)
  • Take 2×10μl samples (put into 96-well plate)
  • Aspirate
  • Add 1ml of buffer A; mix by flipping tube over several times.
  • Spin down (2 minutes at 1500 rpm)
  • Take 2×10μl samples
  • Aspirate
  • Add 1ml of buffer A; mix by flipping tube over several times.
  • Spin down (2 minutes at 1500 rpm)
  • Take 2×10μl samples
  • Aspirate
  • Add 1ml of buffer A; mix by flipping tube over several times.
  • Spin down (2 minutes at 1500 rpm)
  • Take 2×10μl samples
  • Add 200μl of diluted bradford dye to each well
  • Run TECAN absorption at 595nm

4) Set-up experiment in SepCon- to see if IgG unbinds from protein A beads and then diffuses up from the basolateral to the apical of the SepCon

[In the end, we want to keep volume at 1ml: 800μl in bottom, 200μl in top of SepCon, use basolateral to wet backside of membrane]

  • Spin down beads (2 minutes at 1500 rpm)
  • Aspirate
  • Add 500μl of buffer B to centrifuge tube, mix well, put solution into bottom of the transwell (24-well plate)
  • Add 300μl of buffer B to centrifuge tube, mix well, get the rest of the beads
  • Wet back of membrane with buffer in basolateral
  • Add 200μl of buffer B to apical
  • Shake at 200 RPM for ~1 hour (tape down so plate doesn’t rattle)

5) After 1 hour, need to get beads back into a neutral solution (they can’t be at extreme pH’s for too long)

  • Transfer SepCon to a neighboring well
  • Transfer beads into the 2ml tube
  • Spin down (2 minutes at 1500 rpm)

For solution:

  • Transfer solution on top of beads back into the well
  • Replace SepCon, let set-up sit at 0 RPM overnight

For beads:

  • Use buffer A to transfer beads into a 15ml tube
  • Wash with buffer A (~5ml)
  • Store in 20% ethanol (8ml buffer A and 2ml 100% ethanol)

6) Get results

  • Take 2×10μl samples each from the apical and basolateral of SepCon to see if IgG has diffused through the membrane.