SDS-PAGE ANALYSIS OF LIGHT INDUCED PROTEINS

Developed by Kathleen Diehl and Dale Vogelien

Electrophoresis is a technique for separating and characterizing proteins and nucleic acids.
For protein studies, one can use it to show in a semi-quantitative manner how many proteins there
are in a sample, whether two samples differ qualitatively or quantitatively in their protein
composition, and whether a specific protein is present in a sample. One might, for instance, use
electrophoresis to tentatively document changes in protein composition that occur as an organism
progresses from one developmental stage to another, changes in protein composition that occur upon
exposure to a given treatment, and differences in the protein composition of two tissues.
Comparison of "protein profiles" obtained from tissues under different conditions can indicate
similarities (e.g. proteins present in all), differences (e.g. proteins unique to one condition), and
quantitative changes in the level of a given protein. Proteins unique to a particular condition are
potentially useful in ultimately understanding the cellular events that are occurring at that time.
Similarly, a comparison of protein profiles from unstressed and stressed organisms could lead to the
identification of proteins associated directly or indirectly with the stress response. It is important to
realize, however, that electrophoresis indicates potentially interesting differences. It does not, by
itself, give the identity of a protein(s).
Electrophoresis depends on the observation that different proteins are different sizes and
have different overall surface charges at a fixed pH. Differences in size are due to differences in the
number of amino acids that make each protein. Different proteins of similar size, however, will
likely vary in overall charge due to the specific amino acid sequence that each different protein has.
Thus, different proteins can be separated from one another by taking advantage of differences in size
and/or differences in overall surface charge. Electrophoresis, in general, consists of placing a
mixture of charged molecules in a solid supporting medium (e.g. a gel slab), immersing the gel slab
in a buffered salt solution, and establishing an electric field between two electrodes positioned at
both ends of the gel. The charged molecules will migrate towards the positive or negative electrode,
the direction dependent upon their overall charge (e.g. molecules with an overall negative surface
charge will migrate towards the positive electrode). Several variations of electrophoresis are
available for the separation of nucleic acids and proteins. Descriptions of the different kinds of
electrophoresis that can be used to separate proteins are described below.
One kind of electrophoresis uses a starch or agarose gel to separate proteins that are identical
in size (and may even have the same activity), but differ in their overall surface charge because of
amino acid substitutions. Most students have preformed this type of electrophoresis in KSU's
genetics lab when examining different forms of hemoglobin (e.g. adult, fetal and sickle cell
hemoglobins). Each form of hemoglobin was of similar size and had an overall negative charge
(buffer pH 9.2). However, a difference as small as a single amino acid substitution can result in a
significant change in the magnitude of the surface charge. Consequently, when all forms are placed
in an electrical field, all move towards the positive electrode, but at a rate that is determined by the
size of the negative charge (e.g. the greater the overall negative charge the faster the rate of
migration through the gel). Thus, in this case the proteins move at different rates in an electric field
due to differences in overall (negative) surface charge.
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Another type of electrophoresis is PAGE (short for polyacrylamide gel electrophoresis).

Polyacrylamide is a polymer that forms a gel with pores that are relatively small, approximately the
size of proteins. "Native" PAGE is performed under conditions of high pH, a condition which
causes almost all proteins to have overall negative charges. The system separates proteins both by
their surface charge density and their size (larger proteins move more slowly because their ability to
move through the pores of the gel is restricted). The addition of sodium dodecylsulfate (SDS), a
detergent, to the PAGE system led to one of the most widely used techniques in the study of proteins
- SDS-PAGE. SDS coats individual proteins, negating the charge differences due to amino acid
composition. Samples are first heated (to 100 C) in a buffer containing SDS and reducing agents
(e.g. mercaptoethanol or dithiothreitol). These conditions denature polypeptide chains (as well as
separate protein subunits in oligomers). As previously noted, the SDS then surrounds individual
polypeptide chains, giving each chain the same overall surface charge (e.g. all proteins will have the
same net negative charge). Under these conditions, electrophoresis separates proteins by size. The
addition of protein molecular weight standards (a series of proteins of known molecular size) can be
used to determine the relative size of an unknown protein.
In this experiment you will use SDS-PAGE to compare protein composition of shoot tissue
isolated from plants grown in complete darkness to plants grown in the light (under a 12 hour
photoperiod). These two groups of plants are visibly different in appearance. The shoots of dark
grown plants are etiolated (e.g. whitish yellow in color/lacking chloroplasts, very elongated, have
larger internode lengths, smaller leaves, but capable of greening and normal development upon
exposure to light), where as the light grown plants are typical pea plants. Given the morphological
and physiological differences between these two groups, it is expected that they will also differ in
the proteins found in their cells. Some of these proteins will be proteins whose synthesis is directly
induced by the perception of light and associated with the differentiation of etioplasts to
chloroplasts. In addition, you will expose dark grown plants to increasing durations of light to
document the timing of light induction of proteins. If all works well, you should be able to detect
several protein differences between light and dark grown plants, characterize these proteins by
size/weight (kiloDaltons), identify the photosynthetic protein Rubisco in a protein profile by running
a Rubisco standard, and document the timing of Rubisco induction.
Rubisco is a chloroplast specific photosynthetic protein. It is a key enzyme in the light
independent reactions (or Calvin cycle), catalyzing the fixation, or attachment, of atmospheric
carbon dioxide to ribulose 1,5-bisphosphate. The resulting three carbon acid is converted to
phosphoglyceraldehyde, the first sugar to be produced, using ATP and NADPH (products of the
light dependent reactions). Rubisco is an abundant protein, accounting for 40 80 % of all soluble
protein in most leaves (Hopkins, 1999). It is made up of 16 polypeptide chains, including 8 small
subunits (about 14 kDa each) and 8 large subunits (about 55 kDa each), with a molecular weight of
about 560 kDa. The small subunit is coded for by a gene in the nucleus, whereas the large subunit is
coded for by a chloroplast gene. The expression of both genes is influenced by light (Taiz and
Zeiger, 1998; Smith and Ellis, 1981).
The entire study will be performed over three lab periods. The preparation of protein
samples from the various plant treatments will be performed today. The protein concentration in
samples will also be determined at this time. SDS-PAGE, gel staining and analysis will be
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performed during subsequent lab periods.

Procedure
Plant Material (provided by instructor)
The pea plant will be used in this study. Seeds of Alaska Pea have been sown in moist
generic potting mix (prepare 1 flat) and saturated vermiculite (prepare 5 flats). Place the flat
containing soil under cool white fluorescent lights set for a 12 hour photoperiod. The remaining five
trays were placed in complete darkness in a growth chamber. The flat maintained in the light was
watered as needed to prevent drying. Flats grown in the dark did not require additional watering
over the 9 day growth period.
Preparation of Protein Samples From Pea seedlings
An extraction procedure used by Smith and Ellis (1981) for the isolation of soluble
protein from peas will be followed. This extraction procedure is simple and quick. Read
through the procedure before beginning.
1. All glassware and solutions will be provided prechilled and should be kept on ice during the
isolation.
2. Prepare the Extraction Buffer as follows: Transfer 10 ml of 50 mM Tris-HCl, pH 8, with 1 mM
EDTA to a 15 ml centrifuge tube with cap. Add 2 mM phenylmethylsulfonyl fluoride (100 l of
200 mM stock) and 10 mM 2-mercaptoethanol (7 l of commercially available stock). Invert
several times to mix contents.
3. Remove pea leaves (approximately 1 g) from seedlings and place in a chilled mortar. Record
actual fresh weight harvested. NOTE: use the light grown plants for your first extraction
attempt.
4. Add 2 ml of Extraction Buffer and homogenize with pestle until a uniform slurry is obtained.
5. Use a transfer pipet to transfer the slurry to 1 or more microfuge tubes. Try to avoid introducing
air bubbles into the homogenate.
6. Centrifuge in a prechilled microfuge at room temperature for 5 minutes at 14,000 g.
7. Carefully remove supernatant and transfer to a clean microfuge tube, label and store on ice. This
is the protein extract that is to be used in SDS-PAGE analysis. Transfer 100 l of this extract to
another microfuge tube for use in the next step and store this on ice. Store the remaining sample
at 70 C.
8. Repeat the procedure using plants grown in complete darkness, but do not remove the plants
from darkness until you are ready to proceed quickly from start to finish. Also, grind 1 g of leaf
tissue in 1 ml of extraction buffe AND work with room lights off and as little natural light as
possible.
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9. Place the remaining flats of dark grown pea seedlings under light. Extract 1 g of leaf tissue with
2 ml of extraction buffer after 10 and 60 minutes of light exposure. Work with room lights off
and as little natural light as possible.
Estimation of Protein Concentration Using the Coomassie Assay
One of the more rapid, convenient, and frequently used assays for determining protein
concentration is the Coomassie Assay, sometimes called the Bradford Assay. It is based on the
observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250
shifts from 465 nm to 595 nm when the dye binds to a protein. The color is stable for a fairly long
time, which means that the color need not be measured immediately. In addition, the color change is
not affected by the presence of many salts, buffers, and other compounds commonly found in protein
extracts or used in biological experiments. Lastly, the assay can detect as little as 1 g of protein.
1. Turn spectrophotometer on and set the wavelength at 595 nm.
2. Add 1 ml of deionized water to each cuvette you have available.
3. Following the table below, make the following additions of protein standard or sample to
cuvettes. DO NOT WRITE ON CUVETTES. (Note: concentration of protein standard is 0.2
mg/ml; protein used to prepare standard is bovine serum albumin)
vol. of
Tube water
1
100 l
2
95 l
3
85 l
4
50 l
5
25 l
6
0 l
7
98 l
8
90 l
9
98 l
10
90 l
11
98 l
12
90 l
13
98 l
14
90 l

vol. of
standard
total
or sample
g protein
0 l
0
5 l
1
15 l
3
50 l
10
75 l
15
100 l
20
2 l (D)
?
10 l (D)
?
2 l (L)
?
10 l (L)
?
2 l (10T)
?
10 l (10T)
?
2 l (60T)
?
10 l (60T)
?

protein standards

plant extracts

4. Add 2.5 ml of Coomassie Dye Reagent to each tube. The reagent contains a strong acid, so be
careful not to spill any on yourself or equipment.

5. Mix tube contents by holding parafilm over the mouth of tube and inverting a few times. Use a
clean piece of parafilm for each tube.
6. Zero the spectrophotometer using the solution in tube 1 (follow the instructions on the face of
the spectrophotometer).
7. Record the absorbance of the standard solutions (tubes 1 6) in Table 1.
8. Record the absorbances of remaining samples in Table 2. Note: it may be necessary to dilute
samples if absorbance values are outside of standard curve. Similarly, a greater sample volume
may need to be used in protein assay. Note any dilution or change in volume used.
9. Use Excel to prepare a standard curve by graphing O.D. as absorbance at 595 nm (y-axis) vs the
amount of protein (in ug) in the cuvette (x-axis). Use data for tubes 1 - 6 in preparing the
standard curve. Do NOT use other tubes, for they contain sample dilutions.
10. Use the standard curve to determine the amount of protein in each sample. Note: this is the
amount of sample protein in the cuvette (in g). Divide this amount by the volume of sample
added to the cuvette to determine protein concentration in g/l in the original sample. Record
the protein concentration in each sample dilution in Table 2.
Table 1 Data for a Protein (BSA) Standard Curve Using the Coomassie Assay
O.D.
(measured as Abs. @ 595 nm)

Total Protein Concentration of BSA solutions

(g)
0
1
3
10
15
20

Table 2 Protein Concentration for Pea Plants Grown in Darkness and Light

Sample

O.D.
(Abs. @ 595 nm)
2 l
10 l

Protein Concentration
(ug/ul)
2 l
10 l
Avg. l

Dark grown (D)

DG + 10 minute light exposure (10T)
DG + 60 minutes light exposure (60T)
Light grown (L)

Electrophoresis of Samples
1. Obtain and thaw frozen samples. You should have a microfuge tube containing a dark proteins
sample (D), light sample (L) and 2 light exposed treated samples (10T and 60T).
2. Prepare a dilution of each sample with sample loading buffer to achieve a final protein
concentration of 2 ug/ul. Tap tube gently with your finger tip to mix. The sample must be
completely mixed with sample buffer, but try to reduce the amount of liquid that clings to the
walls of the tube.
3. Prepare a 1:10 dilution of the protein molecular weight standard mix with sample loading buffer.
Suggestion: you will load 10l into a well, so prepare 15 l; this means you will need 1.5 l of
the standard mix and 13.5 l of sample loading buffer.
4. Prepare a 1:5 dilution of Rubisco stock solution (a 2.5 g protein/l solution stock is available)
with sample loading buffer. Suggestion: you will load 15 l, so prepare 20 l; use 4 l Rubisco
stock solution and 16 l sample loading buffer.
5. Heat all diluted samples and standard mixes at 95 C for 5 minutes. To do this, set them into a
holder or tray and lower the tray into a beaker of boiling water.
6. While samples are heating, set up the electrophoresis rig. Precast gels (available through BioRad
Laboratories) for the Mini-PROTEAN II and III electrophoresis units will be used. Load the
precast gels into the electrophoresis module by following the instructions provided (refer to
handout at your bench). You will receive a 12% acrylamide gel.
7. Add electrode buffer to the upper buffer chamber (e.g. in between the two gel sandwiches). Fill
until the buffer reaches a level halfway between the short and long plates. Do NOT overfill the
upper buffer chamber, for this will result in siphoning of the buffer over the top of the gasket,
resulting in buffer loss and interruption of the electrophoresis experiment.
8. Pour electrode buffer into the electrophoresis tank so that at least the bottom 1 cm of the gel is
covered. Remove any air bubbles from the bottom of the gel so that good electrical contact is
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achieved. This can be done by swirling the lower buffer with a pipette until the bubbles clear.
9. Now you are ready to load samples into the wells under the electrode buffer. BEFORE YOU
BEGIN, plan how you want to load the samples to make comparisons easiest. A sample loading
plan is given below. At least one well should contain standard molecular weight markers
(indicated in the table below as std) and Rubisco sample (2.5 g/l stock diluted 1:4 with sample
loading buffer). Avoid using the two outermost wells.
well
1
load
D
vol.(ul)
13
protein
(record in ng)

2
10T
13

3
60T
13

4
L
13

5
std
10

6
D
25

7
10T
25

8
60T
25

9
L
25

10
Rubisco
15

Your instructor will show you how to load a sample into the wells if necessary/desired.
10. Place the lid on the top of the gel tank to cover the unit. The correct orientation is made by
matching the colors of the plugs on the lid with the jacks on the inner cooling core. A figure and
instruction sheet for your gel rig and power supply is at your bench.
11. Attach the electrical leads to a power supply. Again, match the colors of the electrical leads with
the colors of the outlets on power pack (consult instruction sheet at your bench if necessary).
12. Turn on the power unit and follow the instruction sheet to set the run for a constant voltage of
200 volts. This is the recommended power condition for optimal resolution with minimal thermal
band distortion. No adjustment of the setting is necessary during the run. The default
limit/setting for A (amps) is acceptable for these power units so there is no need to adjust this.
The usual run time is approximately 35 - 45 minutes (until the dye in the sample solution reaches
the bottom of the gel sandwich). This will depend on individual runs, so DO NOT PROGRAM
A TIMED RUN, rather monitor each gel by observing dye location. Note: the current will
probably read approximately 60 mA per gel (120 mA for two gels) at the beginning of the run.
During the 45 minute run the current will slowly drop to about 30 mA per gel. This drop is
caused by the change in buffer ions in the gel, causing a rise in the resistance in the gel. As one
would expect for Ohms law, V = I*R. At constant voltage (V), a rise in the resistance (R) results
in a drop in the current (I).

Staining of Gels
The staining of proteins in gel slabs should be started immediately after completion of the
electrophoresis run, for proteins will begin to diffuse in the gel when the electrical field is removed.
You will begin this process, but your instructor will complete it for you (simply due to lack of time).
Several staining procedures are available. Coomassie stain, silver stain and heavy metal (Zn)
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stain are often used for protein detection in PAGE gels following electrophoresis. The Coomassie
stain is the most frequently used for protein staining in polyacrylamide gels, largely because fewer
steps are involved and the level of background is negligible. The main disadvantages associated
with common Coomassie stains is that they require a time-consuming and foul smelling methanolacetic acid destaining procedure and are less sensitive in the level of protein detection. The silver
stain has greater sensitivity, requires less time, but involves several time-dependent steps and is very
susceptible to background that interferes with gel analysis.
You will use GelCode Blue Stain Reagent, available from Pierce, for detection of proteins in
your gels. The GelCode Blue Stain is a new Coomassie stain that does not require the cumbersome
destaining steps. The reagent stains only protein, not the gel, resulting in a crystal-clear background
after a Water Wash Enhancement step. This stain is sensitive to 8 ng total protein.
1. After electrophoresis, transfer gel to a clean tray and rinse 3 times, 5 minutes each, with 100 200 ml of deionized water and gentle shaking (use a shaker table or gyratory shaker).
2. Add GelCode Blue Stain Reagent to the tray. Gently shake the tray and periodically check the
protein band development. Staining intensity generally reaches a maximum within 1 hour.
3. Replace the staining reagent with deionized water. Several changes with deionized may be
necessary for optimal results. This water equilibration enhances staining sensitivity as weak
protein bands further develop in deionized water over a period of 1-2 hours.
Analysis of Gels
1. Measure the distance of migration of each standard protein and of the bromphenol blue dye.
Divide the migration distance of each protein by that of the dye to determine an "Rf" value for
each protein standard.
2. Use Excel to plot the logarithm of the molecular weight of each standard protein (the molecular
weight of each protein in the standard is provided by the company upon purchase; this
information will be provided to you at this time) on the y-axis and the corresponding Rf value on
the x-axis. Alternatively, the molecular weights can be plotted directly if the y axis is a log
scale. You now have a calibration curve by which you can calculate the molecular weights of
unknown proteins in your samples - all you need is an Rf value. Note: the molecular weights of
the six proteins in
the standard mix are 97,400 (phosphorylase b), 66,200 (serum albumin), 45,000 (ovalbumin),
31,000 (carbonic anhydrase), 21,500 (trypsin inhibitor protein), and 14,400 (lysozyme) daltons.
Hopefully you were able to detect all of these.
3. Compare protein profiles from dark and light grown plants. Note 1) the 4 most abundant bands
in each profile (record distance migrated from well) and 2) any band differences (note nature of
difference and distance migrated) between these protein profiles. Look for bands present in the
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dark grown plant profile and absent in the light grown plant profile, bands absent in the in the
dark grown plant profile and present in the light gown plant profile, and bands that are present in
both profiles - but at significantly different intensity/level. Determine the Rf value for each of
these proteins.
4. Determine the molecular weights of the above proteins using the calibration curve generated in
step2. This characteristic will be used to refer to these proteins in the future (e.g. ..the 23
kDa protein form dark grown plants..).
5. Determine the Rf value and molecular weight for the subunits in the Rubisco sample. Are any of
the bands noted in step 3 Rubisco subunits? Compare the observed size with the known size of
these subunit. Are you close?
6. Describe the light induction of Rubisco. Are one or both subunits more abundant in the light?
Did you observe light induction for one or both subunits? If yes, how long is exposure to light
required to first detect this protein or to detect levels comparable to that of light grown plants?

Assignment/Questions
1. Provide Table 1 and the standard curve generated with this data. Use Excel to generate the
standard curve, trend line equation, and R2 value. Don't forget to title the graph.
2. Provide Table 2.
a. Show all steps/calculations that were used to convert the absorbance values obtained for
the light grown samples to average protein concentration (in g/l).
b. Show all calculations used to determine the final amount of protein (in g) that was
loaded into each well for all protein samples.
3. Provide a table that contains the molecular weight and corresponding Rf values for the low
molecular protein standards used. Provide the standard curve obtained using this data. Use
Excel to generate the standard curve and provide the line equation.
4. Write a paragraph summarizing your findings for the comparison of protein profiles from light
and dark grown plants (refer to data collected for steps 3 and 4 under Analysis of Gels). Refer to

specific proteins by size (e.g. 14 kDa protein) and note their relative significance (e.g. most
abundant protein in the shoots of dark grown plants, etc.)
5. Write a paragraph summarizing your findings for the light induction of Rubisco. Include in your
paragraph your observed molecular weights for the large and small subunits and their known
molecular weights.
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References
Hopkins, W.G. (1999) Introduction to Plant Physiology (2ond ed.), John Wiley and Sons, Inc.,
New York.
Mohr, H. and P. Schopfer (1995) Plant Physiology, Springer-Verlag, New York.
Sambrook, J., E.F. Fritsch and T. Maniatis (1989) Molecular Cloning (2ond ed.) Cold Spring
Harbor Laboratory Press, New York.
Smith, S. and R.J. Ellis (1981) Light-Stimulated Accumulation Of Transcripts of Nuclear and
Chloroplast Genes for Ribulosebisphosphate Carboxylase. J. Mol. Appl. Genet.1: 127137.
Taiz andSieger (1998) Plant Physiology (2ond ed.), Sinauer Associateds, Inc., Massachusetts.

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