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 harvest9.17
WT 3rd harvest7.42
WT 4th harvest6.74
Wt 5th harvest6

Image:Lab_7_SDS_Baculovirus.jpg‎

Lane Content
1MW marker
22.5 hour (-) WT virus
32.5 hour (+) WT virus
4WT 2nd harvest
5Wt 3rd harvest
6WT 4th Harvest
7WT 5th davest
8TNFa control
92.5 hour (+) Rc virus
10Rc 2nd harvest
11Rc 3rd harvest
12Rc 4th harvest
13Rc 5th harvest
14WT Pellet from 4th harvest
15Rc 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.

Image: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
101000
21090100.096
31585150.126
42080200.162
52575250.217
63070300.251
73565350.299
84060400.325
  • 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)
9LPS2420800.16919.80776
10LPS4820800.14216.45868
11LPS7220800.13415.46636
12IFN gamma2420800.24529.2348
13IFN gamma4820800.26331.46752
14IFN gamma7220800.12814.72212
15LPS+ IFN-g2420800.11513.1096
16LPS+ IFN-g4820800.0839.14032
17LPS+ IFN-g7220800.11913.60576
18Control2420800.26231.34348
19Control4820800.35743.12728
20Control7220800.31738.16568

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


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