User:Eric Ma/Notebook/MICB323 Lab Book/2009/03/24

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Goals

  1. Perform ELISA to detect TNFa in LPS +/- IFNg stimulated culture supernatants.
  2. Perform PAGE on the IP samples from Lab 8
  3. Western Transfer following PAGE


Part 1: ELISA Detection of TNFa in LPS +/- IFNg stimulated culture supernatants.

96 Well Immunoplate Layout

Row Row 1-4 Row 5-8 Row 9-12
A0 pg TNFa (assay buffer only)0 hr PBS controlDay 1 +PBS control
B62.5 pg/mL TNFa30 min +LPS30 min +LPS +IFNg
C125 pg/mL TNFa60 min +LPS60 min +LPS +IFNg
D250 pg/mL TNFa90 min +LPS90 min +LPS +IFNg
E500 pg/mL TNFaSupplied 6 hr +LPS sampleSupplied 6 hr +LPS +IFNg
F1000 pg/mL TNFaSupplied 8 hr +LPS sampleSupplied 8 hr +LPS +IFNg
G2000 pg/mL TNFa1/5 dilution of Day 1 +LPS stimulation1/5 dilution Day 1 +LPS +IFNg
H4000 pg/mL TNFa90 min + IFNgDay 1 +IFNg

Notes

  • Plate incubation time after addition of samples and antibody solution
    • Started at 3:05PM
    • Have to end by 4:00PM when we add in our Streptavidin-HRP, so incubation time will be no longer than 55 minutes rather than the stipulated 1 hr 30 min.
  • Plate incubation time after addition streptavidin-HRP: 25 minutes
    • Did for only 25 minutes. Hopefully there was enough time for binding to occur.
  • Plate incubation time after addition of TMB: 15 minutes
    • Hopefully there is enough time for color formation.
    • After 15 minutes, there wasn't enough color formation. The concentration isn't high enough for an observable reading. We will leave this ON to get color development; Mrs. Hinze will give us the readings tomorrow. NTS: collect the readings tomorrow!!

Part 2: SDS-PAGE

Eric's Gel

Lane Sample ID Sample Content Volume Pipetted (µL)
1PAGE pre-stained MW markers15
2ES1IP 24 hr (+)LPS20
3ES2IP 48 hr (+)LPS20
4ES3IP 72 hr (+)LPS20
5ES7IP 24 hr (+)IFNg20
6ES8IP 48 hr (+)IFNg20
7ES9IP 72 hr (+)IFNg20
8ES4IP 24 hr (+)LPS (+)IFNg20
9ES5IP 48 hr (+)LPS (+)IFNg20
10ES6IP 72 hr (+)LPS (+)IFNg20

Sunny's Gel

Lane Sample ID Sample Content Volume Pipetted (µL)
1PAGE pre-stained MW markers15
2ES10IP 24 hr control20
3ES11IP 48 hr control20
4ES12IP 72 hr control20
5Blue tube48 hr (+)LPS lysate10
6Green tube72 hr (+)LPS Lysate10
7Yellow tube48 hr (+)IFNg Lysate10
8Purple tube72 hr (+)IFNg Lysate10
9Black tube48 hr (+)LPS (+)IFNg lysate 10
10Brown tube72 hr (+)LPS (+)IFNg Lysate10

Notes

  • The gel ran in a funny manner - it was uneven, and resulted in slanting. As the pattern was identical on both my plate and Sunny's plate (i.e. ours were mirror images of each other), we think it may be due to a voltage problem. The wiring may be causing this.
  • As a result, it will be hard for us to measure any sizes of the proteins.

Part 3: Western Transfer

Notes

  • Preparatory steps were done fine.
  • Left with Mrs. Hinze for downstream processing.

Results

ELISA Results

  • The ELISA results (raw data for Tuesday Lab) are here.

Tabluated Average Absorbance Data

Samples Average Absorbance (A450)
0 pg TNFa (assay buffer only)0.12875
62.5 pg/mL TNFa0.141
125 pg/mL TNFa0.1775
250 pg/mL TNFa0.2105
500 pg/mL TNFa0.272
1000 pg/mL TNFa0.40925
2000 pg/mL TNFa0.324
4000 pg/mL TNFa0.95225
0 hr PBS control0.08575
30 min +LPS0.09725
60 min +LPS0.102
90 min +LPS0.10525
Supplied 6 hr +LPS sample0.404
Supplied 8 hr +LPS sample0.13075
1/5 dilution of Day 1 +LPS stimulation0.2265
90 min + IFNg0.081
Day 1 +PBS control0.1085
30 min +LPS +IFNg0.0855
60 min +LPS +IFNg0.09325
90 min +LPS +IFNg0.09925
Supplied 6 hr +LPS +IFNg0.20725
Supplied 8 hr +LPS +IFNg0.17825
1/5 dilution Day 1 +LPS +IFNg0.263
Day 1 +IFNg0.10325

Standard Curve

Calculated Concentration of TNF-alpha

Samples Average Absorbance (A450) 100X Average Absorbance (A450) Calculated [TNFa] (pg/mL) Normalized [TNFa] (pg/mL)
0 hr PBS control0.085758.575-342.3645320
30 min +LPS0.097259.725-285.71428570
60 min +LPS0.10210.2-262.31527090
90 min +LPS0.1052510.525-246.30541870
Supplied 6 hr +LPS sample0.40440.41225.3694581225.369458
Supplied 8 hr +LPS sample0.1307513.075-120.68965520
1/5 dilution of Day 1 +LPS stimulation0.226522.651754.9261081754.926108
90 min + IFNg0.0818.1-365.76354680
Day 1 +PBS control0.108510.85-230.29556650
30 min +LPS +IFNg0.08558.55-343.59605910
60 min +LPS +IFNg0.093259.325-305.41871920
90 min +LPS +IFNg0.099259.925-275.8620690
Supplied 6 hr +LPS +IFNg0.2072520.725256.1576355256.1576355
Supplied 8 hr +LPS +IFNg0.1782517.825113.3004926113.3004926
1/5 dilution Day 1 +LPS +IFNg0.26326.32653.9408872653.940887
Day 1 +IFNg0.1032510.325-256.15763550

TNF-alpha Stimulation Timecourse

Calculations File

Full calculations can be found in this document.


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