User:Eric Ma/Notebook/MICB323 Lab Book/2009/03/10

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Pre-Lab Calculations for SDS-PAGE

  • Note: Unable to do calculations for recombinant virus. Did calculations for Wild-Type virus based on BCA assay results. Did not use BSA Assay results.

Calculations for BSA Assay using WT Virus

Sample Protein Concentration in Original Sample (mg/mL) Volume Needed for SDS-PAGE (µL)
WT 2.5 hr (-) 0.194 15
WT 2.5 hr (+) 0.270 10.76899861
WT 2nd H (+) 0.318 9.166030452
WT 3rd H (+) 0.392 7.420208114
WT 4th H (+) 0.432 6.73912583
WT 5th H (+) 0.486 5.998011079

Calculations for BCA Assay using WT Virus

Sample Protein Concentration in Original Sample (µg/mL) Volume Needed for SDS-PAGE (µL)
WT 2.5 hr (-) virus 1225.8 15.00
WT 2.5 hr (+) virus 1627.6 11.30
WT 2nd harvest + virus 4448.7 4.13
WT 3rd harvest + virus 2750.7 6.68
WT 4th harvest + virus 3926.7 4.68
WT 5th harvest + virus 2064.5 8.91
  • Sunny did an average of the two volumes and pipetted them in.
  • For the recombinant virus samples, we simply pipetted in 15 µL of each sample + 3 µL of 6X loading buffer.

Part 1: Determination of Protein Concentration of LPS-Stimulated Cel Lysates using Bradford Assay

  • Did this together with Sunny.

Standards Prepared

Tube # BSA (1 mg/mL) (µL) Volume H2O (µL)
1 0 100
2 10 90
3 15 85
4 20 80
5 25 75
6 30 70
7 35 65
8 40 60

Lysates Prepared

Tube # Sample Volume of Sample (µL) Volume of H2O (µL)
9 24 hr (+) LPS 20 80
10 48 hr (+) LPS 20 80
11 72 hr (+) LPS 20 80
12 24 hr (+) IFNg 20 80
13 48 hr (+) IFNg 20 80
14 72 hr (+) IFNg 20 80
15 24 hr (+) LPS (+) IFNg 20 80
16 48 hr (+) LPS (+) IFNg 20 80
17 72 hr (+) LPS (+) IFNg 20 80
18 24 hr control 20 80
19 48 hr control 20 80
20 72 hr control 20 80

Part 2: SDS-PAGE of Insect Viral Lysates

  • Prepared two gels: one for myself, one for Sunny.
  • My gel is the gel that had lots of bubbles, and hence not enough wells to load in all the samples.
  • Sunny's gel is perfect. Therefore, Sunny will run the entire 15 samples, while I will omit all of the Recombinant virus lysates.

Lane Guide

Lane Sunny's Gel (Left to Right = Increasing Lane #) Eric's Gel (Right to Left = Increasing Lane #)
1 Pre-stained MW markers Pre-stained MW markers
2 2.5 hr (-) WT virus 2.5 hr (-) WT virus
3 2.5 hr (+) WT virus 2.5 hr (+) WT virus
4 2nd harvest (+) WT virus 2nd harvest (+) WT virus
5 3rd harvest (+) WT virus 3rd harvest (+) WT virus
6 4th harvest (+) WT virus 4th harvest (+) WT virus
7 5th harvest (+) WT virus 5th harvest (+) WT virus
8 TNFa (+) control TNFa (+) control
9 2.5 hr (+) Recombinant virus Polyhedrin pellet WT virus at 3 days p.i.
10 2nd harvest (+) Recombinant virus Polyhedrin pellet recombinant virus at 3 days p.i.
11 3rd harvest (+) Recombinant virus
12 4th harvest (+) Recombinant virus
13 5th harvest (+) Recombinant virus
14 Polyhedrin pellet WT virus at 3 days p.i.
15 Polyhedrin pellet recombinant virus at 3 days p.i.

Notes

  • While running the gel, I noticed that my bands did not migrate down the gel. Therefore, we are discarding my gel. It may have something to do with the bubbles forming in the combs, leading to the stacking gel not forming properly or polymerizing properly.
  • Hence, we only use Sunny's gel for data analysis. My gel was discarded.

Gel Image

090310 SDS-PAGE Cropped gel.jpg

Results of the BSA Assay

Measured Concentrations of the Standards

Tube # BSA (1 mg/mL) (µL) Volume H2O (µL) [BSA] (mg/mL) Absorbance (A595)
1 0 100 0 0
2 10 90 0.1 0.096
3 15 85 0.15 0.126
4 20 80 0.2 0.162
5 25 75 0.25 0.217
6 30 70 0.3 0.251
7 35 65 0.35 0.299
8 40 60 0.4 0.325

Standard Curve

090310 BSA Standard Curve.png

Calculated Protein Concentrations In The Samples

Tube # Sample Volume of Sample (µL) Volume of H2O (µL) Absorbance (A595) [Protein] (mg/mL)
9 24 hr (+) LPS 20 80 0.112 0.130
10 48 hr (+) LPS 20 80 0.098 0.113
11 72 hr (+) LPS 20 80 0.076 0.086
12 24 hr (+) IFNg 20 80 0.204 0.242
13 48 hr (+) IFNg 20 80 0.177 0.210
14 72 hr (+) IFNg 20 80 0.025 0.024
15 24 hr (+) LPS (+) IFNg 20 80 0.08 0.091
16 48 hr (+) LPS (+) IFNg 20 80 0.059 0.066
17 72 hr (+) LPS (+) IFNg 20 80 0.116 0.135
18 24 hr control 20 80 0.208 0.247
19 48 hr control 20 80 0.2 0.238
20 72 hr control 20 80 0.208 0.247

Stimulation Timecourse Graph

090310 Stimulation Timecourse.png

Comments on the trends

  • Stimulation with LPS
    • We see that the stimulation with LPS results in a peak of protein production at 24 hrs p.s. (post stimulation), before gradually decreasing at 48 hrs and 72 hrs p.s.
  • Stimulation with IFN-g
    • We see that the stimulation with IFN-g results in a peak protein production at 24 hrs p.s., gradually decreasing at 48 hrs p.s., and dramatically decreasing at 72 hrs p.s.
  • Combined stimulation with LPS and IFN-g
    • We do not see an observable trend. First, there is a decrease in protein concentration from 24 hrs p.s. to 48 hrs p.s., and then an increase at 72 hrs p.s.

Calculations of RIPA Buffer Volumes

Sample [Protein] (mg/mL) Amount of protein in 350 µL (mg) Volume needed to equal 0.042 mg (µL) Volume of RIPA buffer needed (µL)
24 hr (+) LPS 0.651 0.228 64.9 285.1
48 hr (+) LPS 0.566 0.198 74.7 275.3
72 hr (+) LPS 0.432 0.151 97.9 252.1
24 hr (+) IFNg 1.212 0.424 34.9 315.1
48 hr (+) IFNg 1.048 0.367 40.3 309.7
72 hr (+) IFNg 0.121 0.042 350.0 0.0
24 hr (+) LPS (+) IFNg 0.456 0.160 92.6 257.4
48 hr (+) LPS (+) IFNg 0.328 0.115 128.8 221.2
72 hr (+) LPS (+) IFNg 0.676 0.237 62.5 287.5
24 hr control 1.237 0.433 34.2 315.8
48 hr control 1.188 0.416 35.6 314.4
72 hr control 1.237 0.433 34.2 315.8

Calculations

Full calculations can be found in this file.