Lab 7: S9 cell viability, total protein staining, and [protein] of LPS Macrophage
SDS PAGE of Baculovirus Harvest
- Amount of sample to be used in SDS page according to result from Bradford assay
| Sample
|
Vol of sample for SDS
|
| WT 2.5hr (-) |
15
|
| WT 2.5hr (+) |
10.77
|
| WT 2nd harvest |
9.17
|
| WT 3rd harvest |
7.42
|
| WT 4th harvest |
6.74
|
| Wt 5th harvest |
6
|
| Lane
|
Content
|
| 1 |
MW marker
|
| 2 |
2.5 hour (-) WT virus
|
| 3 |
2.5 hour (+) WT virus
|
| 4 |
WT 2nd harvest
|
| 5 |
Wt 3rd harvest
|
| 6 |
WT 4th Harvest
|
| 7 |
WT 5th davest
|
| 8 |
TNFa control
|
| 9 |
2.5 hour (+) Rc virus
|
| 10 |
Rc 2nd harvest
|
| 11 |
Rc 3rd harvest
|
| 12 |
Rc 4th harvest
|
| 13 |
Rc 5th harvest
|
| 14 |
WT Pellet from 4th harvest
|
| 15 |
Rc Pellet from 4th harvest
|
- we just used 15uL of samples into the sds page for recombinant as none of the protein assay had reliable result
Stimulated Macrophage [protein]
standard curve:
- [protein] in 350uL of lysate.
- this is from the lowest protein concentration.
Figure 1. BSA Standard Curve
Table 1. BSA standard data.
| tube
|
BSA
|
water
|
BSA mass
|
A595
|
|
(uL) |
|
ug |
|
| 1 |
0 |
100 |
0 |
|
| 2 |
10 |
90 |
10 |
0.096
|
| 3 |
15 |
85 |
15 |
0.126
|
| 4 |
20 |
80 |
20 |
0.162
|
| 5 |
25 |
75 |
25 |
0.217
|
| 6 |
30 |
70 |
30 |
0.251
|
| 7 |
35 |
65 |
35 |
0.299
|
| 8 |
40 |
60 |
40 |
0.325
|
- BSA standard was incubated for 10minutes.
- Data is from BSA Standards Eric M. and Sunny D prepared
table 2. Macrophage lysate data.
| tube
|
Treatment
|
Hours incubated
|
vol sample(uL)
|
vol water(uL)
|
A595
|
protein mass (ug)
|
| 9 |
LPS |
24 |
20 |
80 |
0.169 |
19.80776
|
| 10 |
LPS |
48 |
20 |
80 |
0.142 |
16.45868
|
| 11 |
LPS |
72 |
20 |
80 |
0.134 |
15.46636
|
| 12 |
IFN gamma |
24 |
20 |
80 |
0.245 |
29.2348
|
| 13 |
IFN gamma |
48 |
20 |
80 |
0.263 |
31.46752
|
| 14 |
IFN gamma |
72 |
20 |
80 |
0.128 |
14.72212
|
| 15 |
LPS+ IFN-g |
24 |
20 |
80 |
0.115 |
13.1096
|
| 16 |
LPS+ IFN-g |
48 |
20 |
80 |
0.083 |
9.14032
|
| 17 |
LPS+ IFN-g |
72 |
20 |
80 |
0.119 |
13.60576
|
| 18 |
Control |
24 |
20 |
80 |
0.262 |
31.34348
|
| 19 |
Control |
48 |
20 |
80 |
0.357 |
43.12728
|
| 20 |
Control |
72 |
20 |
80 |
0.317 |
38.16568
|
|
|
total protein staining
- heated at 92oC for 3 minutes
Post-lab
- The Bradford for the stimulated Macrophage went well. Result were not as expected.
- the effect of SDS were really shown in this lab when protein concentration were needed.
- S9 Viability was not done
|
Project name
Main project page
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