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.