Test Run of SepCon Format

SepCon Experiment (Carrie): Does it work in a SepCon format?

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

9/29/09

Make Buffer B at pH 3

9/30/09

1) Rinse off ethanol

2) Add IgG

• Take absorbance reading of 1 aliquot of IgG (2×10μl samples) [D7= 0.565abs, D8=0.63abs]
• Transfer beads to 2ml centrifuge tube
• Add human IgG
• Mix (start:4:35pm)
• [Barrett removed later that evening and put it in the refrigerator overnight]

10/1/09

1) Rinse human IgG-protein A beads mixture with buffer A, to remove any unbound IgG

• Take 2×10μl samples [E1&2]
• Take 2×10μl samples [F1&2]
• Take 2×10μl samples [G1&2]
• Take 2×10μl samples [H1&2]
• Add 200μl of diluted bradford dye to each well

TECAN results:

0.2027	0.1985	after spinning down
0.213	0.2055	after 1 rinse with buffer A
0.2156	0.2118	after 2 rinses
0.2072	0.2014	after 3 rinses

2) Make Transwell: Wafer B014 (one pinhole)

3) Conduct experiment in SepCon (See protocol)

After 1 hour, remove protein A beads into neutral experiment, but let buffer B solution run overnight

–> Question: Did the membrane break? Looking at it, there didn’t seem to be any tears, even when I looked at it under the optical microscope. However, when I went to replace the SepCon into the first well, I couldn’t get a bead of buffer B to form on the underside of the membrane.

10/2/09

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

TECAN:

• Apical [F3&4]: 0.337 and 0.348 abs
• Basolateral [G3&4]: 0.333 and 0.361 abs

Conclusions:

• It seems like the human IgG was able to diffuse upward through the membrane because the absorbance readings from both the apical and basolateral are very close.