User:Mark X. Ling/Notebook/Micb 323/2009/03/10

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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

Lab 7 SDS Baculovirus.jpg

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.

MXL Micb323 Lab 7 BSA standard.jpg
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