| Lysates (CL) |
R10H020 |
R10H021 |
R10H022 |
R10H023 |
| |
D7 |
D11 |
D15 |
D7 |
D11 |
D15 |
D7 |
D11 |
D15 |
D7 |
D11 |
D15 |
| Tubulin a-1B |
18 |
17 |
18 |
20 |
19 |
16 |
16 |
20 |
16 |
21 |
19 |
16 |
| Tubulin-b |
20 |
23 |
20 |
23 |
21 |
21 |
19 |
23 |
20 |
24 |
23 |
18 |
| Tubulin b-2C |
18 |
21 |
16 |
22 |
21 |
19 |
18 |
22 |
18 |
23 |
22 |
16 |
| actin-b |
20 |
21 |
21 |
24 |
22 |
23 |
22 |
19 |
16 |
21 |
22 |
13 |
| Gapdh |
17 |
20 |
19 |
20 |
22 |
20 |
16 |
19 |
17 |
20 |
19 |
14 |
| H-ch |
19 |
21 |
23 |
20 |
21 |
21 |
20 |
20 |
20 |
22 |
20 |
20 |
| L-ch |
9 |
11 |
12 |
11 |
12 |
12 |
12 |
11 |
11 |
12 |
12 |
10 |
| Sum |
121 |
134 |
129 |
140 |
138 |
132 |
123 |
134 |
118 |
143 |
137 |
107 |
| Norm. Factor |
1 |
0.903 |
0.938 |
0.8643 |
0.8768 |
0.9167 |
0.9837 |
0.903 |
1.0254 |
0.8462 |
0.8832 |
1.1308 |
| Supernatants(SP) |
R10H020 |
R10H021 |
R10H022 |
R10H023 |
| |
D7 |
D11 |
D15 |
D7 |
D11 |
D15 |
D7 |
D11 |
D15 |
D7 |
D11 |
D15 |
| H-ch |
36 |
40 |
41 |
36 |
37 |
42 |
38 |
40 |
41 |
40 |
38 |
42 |
| L-ch |
23 |
20 |
22 |
16 |
20 |
21 |
16 |
21 |
21 |
21 |
19 |
21 |
| Sum |
59 |
60 |
63 |
52 |
57 |
63 |
54 |
61 |
62 |
61 |
57 |
63 |
| Norm. Factor |
1 |
0.9833 |
0.9365 |
1.1346 |
1.0351 |
0.9365 |
1.0926 |
0.9672 |
0.9516 |
0.9672 |
1.035 |
0.937 |
The peptide count of these selected proteins in each sample was summed up, and the sum obtained from H20D7_CL and H20D7_SP samples (121 and 59, respectively),
were arbitrarily set as normalization standard, i.e., possessing a normalization factor (NF) of 1. Normalization of the proteins in other samples was accomplished by dividing
the total peptide counts of these house-keeping proteins by that of H20D7_CL or H20D7_SP, for samples derived from cell lysates or culture supernatants, respectively.
House-keeping proteins used for CL samples included glyceraldehyde-3-phosphate dehydrogenase, actin β, tubulin β, tubulin α-1β and tubulin β-2C. Additionally, the H-ch
and the L-ch of the therapeutic protein being expressed by the cell were included in this normalization process. For the data generated from SP samples, the normalization
process used only the H-ch and L-ch of the therapeutic ab being expressed by the cell line. This was necessary as the above house-keeping genes were either undetectable
or present in very low levels in the SP samples. Even though the H-ch and L-ch protein bands were largely avoided during the sample preparation step (described above
in the sample preparation section), sufficient quantities of both of these proteins were still retained in the sample to justify using them as housekeeping genes. |
