Digication ePortfolio :: BCH403FIELDS :: Week 2: Isolation of Protein for SDS-PAGE Analysis

DRAFT: This module has unpublished changes.

Week 2: Isolation of Protein for SDS-PAGE Analysis

Monday, November 14, 2011

Group 3 (Tori, Samantha, Stephanie, Jared, Karafaye)

Purpose

The purpose of this week's experiment is to isolate the protein for each of the plants and then run an SDS page gel electrophoresis in order to perform a Western Blot analysis.

Introduction

Using the leaves of each of the Wisconsin Fast Plants, protein will be isolated and an assay will be performed. The assay will help to determine which proteins are present in which species of plant being studied. SDS-PAGE electrophoresis will be performed in order to carry out a Western Blot analysis in week three.

Procedural Outline

Isolation of Protein for SDS-PAGE

Label eight microfuge tubes (one corresponding to each plant).
Create eight pestles (one for each plant) with a blue pipet tip by melting the tip with a flame (from a match) and pressing it against a sterile surface to create a rounded point.
Grind one leaf from each plant in the microfuge tubes with the pestle.
Place each tube on ice with 1 mL of QB and then spin it at top speed in the microfuge at 4˚ C for 15 minutes.
Transfer the liquid supernatant into a second microfuge tube.
With the supernatants, make three dilutions of 1:2, 1:20, and 1:200 in a new microfuge tube for a 100µl final volume using QB buffer.
Make three different dilutions of unknowns with 1:2, 1:20 and 1:200 dilution factors.

Assay and Electrophoresis

Label six microcentrifuge tubes 1-6 along with the protein determination reagent of 200µl of copper II sulfate pentahydrate solution and 10 mL of bicinchoninic acid.
Prepare the standards with the indicated amounts of buffer, protein standard, and unknown protein samples in appropriate microfuge tubes based on the following table:

Tube #	Dilution Factor	QB Buffer (microliters)	Progtein Standard (microliters)	Total (Microliters)
1	-	100	0.0	100
2	-	80	20	100
3	-	60	40	100
4	-	40	60	100
5	-	20	80	100
6	-	0.0	100	100
	1:2	X	X	100
	1:20	X	X	100
	1:200	X	X	100

Once the protein standards and unknowns are made, vortex the samples immediately.
After vortexing, load ten microliters of the samples into 96-well plate along with 200 microliters of protein determination reagent.
Incubate the well for 30 minutes at 37 degrees C.
Plot a standard curve for the average protein standard concentration vs. the net absorbance values at 560 nm along with the linear regression and repot of slope, intercept, and R squared values.
For the electrophoresis procedure, use QB buffer to dilute out quantified samples to the same concentration as the concentrated sample using the C1V1=C2V2 formula.
Denature the samples at 95 degrees C for 10 minutes and mix with 30 microliters of diluted protein sample with 15 microliters of loading buffer.
Load as many samples as possible in the gel based on the following confuguration: M1a, M2a, M3a, M4a, and M1b, M2b, M3b, M4b.
Run the gel at 100 V for one hour wrapped still between the glass in a plastic bag with a few mL of gel running buffer.

Results

Stem Length (mm)
	DAYS	1	2	3	4	5	6
Variegated	1a	29	29	35	38	42	43
Variegated	1b	18	18	18	24	25	27
Purple hairy	2a	14	18	33	33	38	41
Purple hairy	2b	40	47	58	61	66	56
Standard	3a	26	26	27	30	35	36
Standard	3b	35	40	51	54	58	59
Hairless non purple	4a	21	23	24	32	35	36
Hairless non purple	4b	26	30	32	35	49	55

Table One: Stem Lengths. This table displays the stem length for each Brassica rapa plant measured over the six days.

DAY 2	leaf 1 (mm2)	leaf 2 (mm2)	leaf avg. (mm2)
1a	36	15	25.5
1b	21	8	14.5
2a	40	18	29
2b	50	24	37
3a	50	36	43
3b	40	15	27.5
4a	18	14	16
4b	21	24	22.5

Table Two: Day Two Results. This table displays the measurements of the leaf areas measured in millimeters squared on day two.

DAY 3	Leaf 1 (mm2)	Leaf 2 (mm2)	Leaf Avg (mm2)
1a	45	24	34.5
1b	21	18	19.5
2a	60	45	52.5
2b	96	60	78
3a	88	77	82.5
3b	70	35	52.5
4a	36	24	30
4b	50	43	46.5

Table Three: Day Three Results. This table displays the measurements of the leaf areas measured in millimeters squared on day three.

DAY 4	Leaf 1 (mm2)	Leaf 2 (mm2)	Leaf avg (mm2)
1a	40	60	50
1b	45	24	34.5
2a	77	60	68.5
2b	112	72	92
3a	108	96	102
3b	88	50	69
4a	54	60	57
4b	66	66	66

Table Four: Day Four Results. This table displays the measurements of the leaf areas measured in millimeters squared on day four.

DAY 5	Leaf 1 (mm2)	Leaf 2 (mm2)	Leaf Avg (mm2)
1a	50	57	53.5
1b	20	45	32.5
2a	63	84	73.5
2b	108	90	99
3a	150	126	138
3b	70	96	83
4a	66	54	60
4b	43	90	66.5

Table Five: Day Five Results. This table displays the measurements of the leaf areas measured in millimeters squared on day five.

DAY 6	Leaf 1 (mm2)	Leaf 2 (mm2)	Leaf Avg (mm2)
1a	98	77	87.5
1b	36	24	30
2a	126	104	115
2b	160	96	128
3a	125	165	145
3b	140	96	118
4a	130	140	135
4b	140	150	145

Table Six: Day Six Results. This table displays the measurements of the leaf areas measured in millimeters squared on day six.

Figure One: Stem Growth by Day. This graph represents the growth of the stems of the Brassica rapa plants from day one to day six. Plants 1a and 1b, (dark blue and maroon), are variegated Brassica rapa. Plants 2a and 2b (green and dark purple), are hairy purple stem Brassica. Plants 3a and 3b, (light blue and orange), are standard Brassica rapa. Plants 4a and 4b, (periwinkle blue and red), are hairless non purple stem Brassica rapa.

Figure Two: Stem growth by Day Average. This graph represents the growth of the Brassica rapa stems from day one to day six. Because we plotted two of each type of plant, each of the same plants were averaged in order to compare it to the stem growth of each. Plant 1, dark blue, is variegated Brassica rapa. Plant 2, red, is a hairy purple stem Brassica rapa. Plant 3, green, is a standard Brassica rapa. Plant 4, orange, is a hairless non purple stem Brassica rapa.

Figure Three: Leaf Growth by Day. This graph represents the growth of Brassica rapa leaves from day two to day six. Plants 1a and 1b, (dark blue and maroon), are variegated Brassica rapas. Plants 2a and 2b, (green and dark purple), are hairy purple stem Brassica rapas. Plants 3a and 3b, (light blue and orange), are standard Brassica rapa. Plants 4a and 4b (periwinkle blue and red), are hairless non purple stem Brassica rapa.

Figure Four: Leaf Growth by Day Average. This graph displays the growth of Brassica rapa leaves from day two to day six. Each different plant was plotted twice in order to observe a growth different in each. Therefore, the values were averaged in order to process the difference. Plant 1, dark blue, is variegated Brassica rapa. Plant 2, red, is a hairy purple stem Brassica rapa. Plant 3, green, is a standard Brassica rapa. Plant 4, orange, is a hairless non purple stem Brassica.

Averages of the Plant Species Stem Lengths (mm2)
	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1a and 1b	23.5	23.5	26.5	31	33.5	35
2a and 2b	27	32.5	47	47	52	48.5
3a and 3b	30.5	33	39	42	46.5	47.5
4a and 4b	23.5	26.5	28	33.5	42	45.5

Table Seven: Averages. This table displays the average lengths of each plant stem that were calculated for each day in order to perform the T Test.

Conclusion

Protein extraction and SDS-PAGE gel electrophoresis was successfully carried out in order to perform a Western Blot analysis for week three.

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