Run | O.D
488 nm
a)
| EGFP conc. (mg/ml)
b)
| Recovery (%)
c)
| Retention Time (min) | Peak Area | O.D
488 nm
d)
| Retention Time (min) | Peak Area |
---|
Unbound fraction
|
Bound fraction
|
20
| 0.318 | 0.288 | 96.108 | 17.204 | 41318386 | 0.042 | 35.119 | 21663431 |
40
| 0.31 | 0.28 | 93.291 | 17.198 | 41864592 | 0.042 | 35.132 | 20405735 |
60
| 0.316 | 0.286 | 95.404 | 17.13 | 46612910 | 0.043 | 35.003 | 27522817 |
80
| 0.314 | 0.284 | 94.7 | 17.234 | 48029530 | 0.043 | 35.087 | 40458786 |
100
| 0.313 | 0.283 | 94.348 | 17.116 | 47410534 | 0.042 | 34.888 | 29504690 |
120
| 0.302 | 0.271 | 90.474 | 17.117 | 42958986 | 0.042 | 34.9 | 24795107 |
140
| 0.309 | 0.279 | 92.939 | 17.157 | 44253260 | 0.042 | 34.918 | 23585379 |
160
| 0.312 | 0.282 | 93.995 | 17.143 | 37695839 | 0.043 | 34.889 | 21280293 |
180
| 0.314 | 0.284 | 94.7 | 17.119 | 38865664 | 0.042 | 34.906 | 21914998 |
200
| 0.317 | 0.287 | 95.756 | 17.166 | 39476099 | 0.042 | 34.916 | 25579936 |
Average
|
0.313
|
0.283
|
94.172
|
17.158
|
42848580
|
0.042
|
34.976
|
25671117.2
|
S.D. |
0.0047
|
0.0049
|
1.6456
|
0.042
|
3665255.437
|
0.0005
|
0.1
|
5954454.913
|
C.V.(%)
|
1.502
|
1.731
|
1.747
|
0.245
|
8.554
|
1.19
|
0.286
|
23.195
|
- a) The unbound fractions were concentrated to 1 ml, for which the UV absorbance was measured at 488 nm.
- b) EGFP concentration was calculated using the standard curve, as shown in Figure 3H.
- c) EGFP recovery (%) = EGFP concentration/0.3 × 100.
- d) The bound fractions were concentrated to 1 ml, for which the UV absorbance was measured at 488 nm.
- EGFP-spiked plasma (300 μg of EGFP and 2.62 mg of plasma in 100 μl) was depleted every 20th run from runs 20 to 200. Ten chromatograms were analyzed to calculate retention time and peak area. At the same time, unbound fraction and bound fraction from 10 independent depletions were concentrated to 1.0 ml, and OD488 nm was measured to calculate recovery. Concentrations of EGFP in the unbound fraction and bound fraction were calculated according to the standard curve, generated in Figure 3. The coefficient of variation (C.V. (%)) was calculated from the standard deviation, divided by the average and multiplied by 100. There was fluctuation in the 4th run, causing increase in C.V. (%) in the bound fraction peak area.