DRAFT: This module has unpublished changes.

Week 2: Isolation and Quantification of Brassica Protein

 for SDS-PAGE Analysis

Monday, November 14th, 2011

 

Purpose

The purpose of this week's experiment is to isolate and quantify the protein from each of the plants' leaves and then to run an SDS-PAGE gel electrophoresis in preparation for conducting a Western Blot analysis during week III.


 

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 III.

   

Procedure

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

100

 

1:20

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. 

Discussion and Conclusion

Protein obtained from the leaves of the Brassica plants were successfully qunatified, and the gel that was run during this laboratory period will be stored appropriately until next lab meeting, where a Western Blot analysis will be conducted. This laboratory notebook laso provides the phenotypical data measured by all different groups for thier own plants exposed to differeing LED light regimens, as well as an artificial sunlight Greenhouse control group. Percent growh was calculated amongst all of the plants exposed to differing intensities of light within each Brassica genoype. T-tests were performed to demonstrate whether the percent growth calculated from the first dayt of preliminary data collection to the  last day of data collection were significant.

 

Based on the phenotypic observations, the four strains of Brassica rapa grew as expected when exposed to fluorescent light. Each plant’s growth by day was observed and compared to other plants exposed to different light sources. However, our plants had a 50% increase in growth over the week. Within this experiment, we were able to compare the effects of different light spectrums on plant growth, focusing on red light, as well as the biochemical and phenotypic aspects of Brassica rapa.  Light is perceived as three distinct types of photoreceptors: phytochromes,, cryptochromes, and phototropins.  While phytochromes mainly perceive red and far-red light, cryptochromes and phototropins recognize ultraviolet and blue light (Takano et al 2009).  Phytochrome is translocated from the cytoplasm to the nucleus in response to a red light pulse that photoconverts the red absorbing form (Pr, or phytochrome A) of phytochrome into the active far-red absorbing pytochrome (Pfr, or phytochrome B). While phytochromes mainly perceive red and far-red light, cryptochromes and phototropins recognize ultraviolet and blue light (Takano et al 2009). The direct interaction of Pfr in the nucleus with transcription factors is an mportant step in phytochrome regulation of hypocotyls elongation (Nozue 2006). The results were expected to produce an uneven ratio of phytochrome A to phytochrome B in the western blots. Higher ratios of phytochrome B should have been present due to the higher intensity of red light exposed to the Brassica rapa plants. These light treatments respond by inducing germination, chloroplast development, leaf expansion, regulation of gene expression, inhibition of cell elongation, and photoperiodic control of flowering (Reed, 1994). Despite the poor readings in the western blot, we were still able to observe the effects of phytochrome A and B on the Brassica rapa plants from our phenotypic results. Throughout the week, we observed a substantial increase in the stem length and surface area of the leaves each day. Based on these observations, we can suggest that our western blots would show considerably higher levels of phytochrome within the Brassica rapa plants.

In comparison to the other groups, our Brassica rapa plants' growth under red light within the week was only a 50% increase as the other light spectrums demonstrated over 100% increase in percent growth. Variegated plants exposed to LED and infrared light had the most significant percent growth at 2.5 times its original height for the variegated stem and red light had the smallest percent growth, only increasing by one-half of its original height (refer to Figure 3). Plants with purple hairy stems exposed to blue light increased by almost three times its original height whereas far-red had the least amount of growth by percent (refer to Figure 4).  The standard plants under LED light significantly increased in percent growth by nearly four times its original height, and standard plants exposed to red light increased by the smallest percent growth of only one-half of its original height (refer to Figure 5).  Non-purple/non-hairy plants exposed to artificial sunlight grew 4.5 times its original height and non-purple/non-hairy plants exposed to red light almost doubled  its original height, which was the smallest percent growth (refer to Figure 6).  Based on the expected results and hypothesis, the Brassica rapa plants exposed to red light should have shown a more substantial increase in growth in addition to far-red light.

DRAFT: This module has unpublished changes.