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

Revision as of 15:18, 24 March 2009

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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.

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

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

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

@@ Line 151: / Line 151: @@
 * Hence, we only use Sunny's gel for data analysis. My gel was discarded.
-==Results==
+==Results of the BSA Assay==
-===Part 1: BSA Assay===
+===Measured Concentrations of the Standards===
 {| {{table}}
 | align="center" style="background:#f0f0f0;"|'''Tube #'''
@@ Line 177: / Line 177: @@
 |}
-* Standard Curve:
+===Standard Curve===
 [[Image:090310_BSA_Standard_Curve.png|800px]]
+===Calculated Protein Concentrations In The Samples===
 {| {{table}}
 | align="center" style="background:#f0f0f0;"|'''Tube #'''
@@ Line 212: / Line 213: @@
 |}
+===Stimulation Timecourse Graph===
 [[Image:090310_Stimulation_Timecourse.png|800px]]
-* Comments on the trends:
+===Comments on the trends===
-** Stimulation with LPS
+* 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.
+** 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
+* 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.
+** 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
+* 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.
+** 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.
-<br>
-*Calculations of RIPA Buffer Volumes
+===Calculations of RIPA Buffer Volumes===
 {| {{table}}
 | align="center" style="background:#f0f0f0;"|'''Sample'''