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Complete Chemicals and Instructions.

Description

Cyanobactera, also known as blue-green algae, obtain their energy by photosynthesis using sunlight as their energy source. These organisms have been considered to be the oldest and the most important bacteria on the earth. It is believed that they were responsible for the initial oxygenation of the earth’s atmosphere through photosynthesis and it is also felt they were the precursors to the chloroplasts that are found in true algae and plants. There are two classes of photosynthetic pigments in Cyanobactera. The first class contains water-soluble proteins and the major protein is called Phycocyanin, which is blue. The other classes of photosynthetic pigments that include the carotinoids and chlorophylls are small molecular weight molecules and are insoluble in water but soluble in organic solvents such as alcohol. In this laboratory exercise, 8 groups of students isolate and characterize both groups of pigments. In part A of this exercise, students prepare a water-soluble extract from blue green algae and show that it contains the single major protein Phycocyanin by electrophoresis as shown in the gel below. They also determine the charge of this protein by comparing its electrophoretic mobility to the mobilities of dyes with known charges. In part B, they prepare an alcohol extract and analyze the smaller alcohol soluble pigments by thin layer chromatography in order to identify the chlorophylls and major carotenoid pigments. The results of this two-part study give students practical hands-on experience with isolation of components from cells as well as electrophoresis and thin layer chromatography and introduces them to one of the most important organisms on the earth.

Contents

Contents included:
· Complete Instructions
· Blue –Green Algae
· Dye Sample 2
· Dye Sample 2
· Glycerol Solution
· Thin-Layer Sheet
· Chromatographic Tubes
· 1.5 ml Tubes
· 1ml Transfer Pipits
· Small Pipits

Requirements

Accessories and Chemicals Needed:
· Electrophoresis Equipment
· Automatic Pipetors (Optional)
· Methanol

Sample

Sample from the Student Guide.

Electrophoretic and Chromatographic Analysis of Photosynthetic Pigments from Blue- Green Algae
Background Information

1. Cyanobactera

Cyanobactera, also known as blue-green algae or blue green bacteria, obtain their energy by photosynthesis using sunlight as their energy source. These organisms have been considered to be the oldest and the most important bacteria on the earth. It is believed that they were responsible for the initial oxygenation of the earth’s atmosphere through photosynthesis. Today, cyanobacteria are found in freshwater and in the oceans and are responsible for the production of 20–30% of the Earth’s oxygen. It is also felt that these bacteria were the precursors to the chloroplasts that are found in true algae and plants.
There are two classes of photosynthetic pigments in Cyanobactera. The first class are the water-soluble proteins and the major protein is called phycocyanin, which is blue. This protein traps sun energy and transfers it to chlorophyll. The other class of photosynthetic pigments that include the carotenoid and the chlorophylls are small molecular weight organic (carbon containing) molecules and are insoluble in water but soluble in organic solvents such as alcohol. Chlorophyll a, the major chlorophyll type, traps the energy of the sun to make carbohydrate during the process of photosynthesis. The carotenoid pigments, which are generally orange or yellow also pass adsorbed energy to chlorophyll a and consequently assist in photosynthesis. An example of a carotenoid is beta-carotene, which is responsible for the orange color of carrots. Table 1 lists some properties of these photosynthetic pigments and Figure 1 shows diagrams of the chemical structures of chlorophyll a and beta-carotene. In this laboratory exercise, you will isolate and characterize both groups of pigments.

Table 1. Characteristics of the Photosynthetic Pigments in Cyanobactera
Pigment Color Chemical Nature Water Soluble Alcohol Soluble

Phycocyanin Blue Large Protein (MW= 232,000) Yes No

Chlorophyll a Green Small Organic Compound No Yes

Carotenoids

Carotene Orange Small Organic Compound No Yes

Xanthophyll Yellow Small Organic Compound No Yes

Figure 1. Chemical Structures of Chlorophyll a and Beta Carotene
Chlorophyll a Beta-Carotene

2. Thin Layer Chromatography
In 1906, the Russian botanist Tswell introduced the term chromatography (“to write with color”) as a result of his studies on the separation of plant pigments using a column of calcium carbonate. Chromatography, like many other important discoveries, remained largely unnoticed for almost one-half century but in the past 50 years has become one of the most important tools for the rapid separation of biological molecules.
Paper chromatography and thin layer chromatography are used to separate small molecules such as pigments, amino acids, sugars, and dyes. In a common form of this technique, the sample is applied as a spot onto a sheet of adsorbent material and a solvent is allowed to flow through the sheet from one edge. As the solvent travels across the adsorbent material by capillary action, it picks up those sample molecules that are soluble in it and not bound to the adsorbent material. Consequently, sample molecules that move the fastest are those that are most soluble in the solvent and have the lowest affinity for the adsorbent paper. The major difference between paper chromatography and thin layer chromatography is the nature of the adsorbent sheet used in the separation. With paper chromatography, the sheet is adsorbent paper such as filter paper. In contrast, thin layer chromatography is performed on a sheet of plastic, or glass, which is coated with a thin layer of adsorbent material such as silica or cellulose. In both types of chromatography, the adsorbent material is known as the stationary phase while the solvent is called the mobile phase. In this laboratory, you will use thin layer chromatography to separate the alcohol soluble pigments that you extract from Cyanobactera. A simple optional experiment, described in Table 1, illustrates the separation of the components of ink by paper chromatography.

Table 2. Separation of the Components of Writing Ink by Paper Chromatography.
(An Optional Experiment)
1. Place samples of different inks as small spots along the shorter edge of a piece of Whatman No. I filter paper (20×30 cm), 2 cm from the edge.
2. After the samples have dried, staple the two longer edges together to form a cylinder.
3. Place the end of the cylinder containing the samples in a jar containing I cm of water. As the water rises, the different colored components of the inks will migrate at different speeds, which are related to their solubility in water and adsorption to the paper.
4. You may wish to compare the results that you obtain to results obtain with a different mobile phase such as 50 % or 100 % alcohol (methanol, ethanol or isopropanol) since some ink pigments are more soluble in alcohol as compared to water.
3. Protein Electrophoresis
Many different types of biological molecules are charged at neutral pH. For example, four of the twenty amino acids found in proteins are charged. The basic amino acids lysine and arginine carry a positive charge while the acidic amino acids aspartate and glutamate carry a negative one. Likewise synthetic dyes, which are similar in size to amino acids, are often acidic or basic. These dyes are commonly used to stain tissue sections, as food coloring agents and for coloring fabrics in the clothing industry. Proteins are composed of amino acids and thus basic proteins are generally rich in lysine and arginine and deficient in aspartate and glutamate while the reverse is true for acidic proteins.
Electrophoresis is the movement of charged molecules under the influence of an electric field. Since many biological molecules carry a charge, they can be separated by this technique. A diagram of the essential components of an agarose electrophoretic system is shown in Figure 2. The agarose gel, containing preformed sample wells, is submerged in buffer, which is contained within the electrophoretic chamber. Samples to be separated are then loaded into the sample wells of the gel. Current from the power supply travels to the negative electrode (cathode), supplying electrons to the conductive buffer solution, gel, and positive electrode (anode), thus completing the circuit.
Agarose is a natural polysaccharide of galactose and anhydrogalaclose derived from agar, which in turn is obtained from certain marine red algae. The agarose gel is an ideal solid support for the separation of proteins and smaller dyes and amino acids on the basis of charge and for the separation of DNA fragments on the basis of size as illustrated in Figure 2. The pore size of a low percentage agarose gel (1%) is much larger than the size of amino acids, dyes and proteins. Thus, the gel can be viewed as a sponge, which has pores large enough to allow even the largest proteins to pass unimpeded. Under these conditions, acidic amino acids (glutamic acid and aspartic acid), acidic dyes and acidic proteins (which have high concentrations of acidic amino acid residues) migrate toward the positive electrode and basic amino acids (lysine acid and arginine), basic dyes and basic proteins (which have high concentrations of basic amino acid residues) migrate toward the negative electrode. The pH of the electrophoresis buffer that you will use in this experiment is 8.6. At this pH, nearly all proteins and dyes have a net negative charge and might toward the positive electrode during agarose gel electrophoresis. The rate of their migration is directly related to ratio of acidic charges/basic charges and consequently proteins with a high ratio of acidic /basic amino acid residues might faster than proteins with a lower ratio of acidic /basic residues.
Figure 2. Components of a Horizontal Electrophoresis System.

4. Proteins Used as Standards in this Study
Hemoglobin – Hemoglobin is the major protein found in red blood cells. This protein transports oxygen from lungs to tissues. The isoelectric point of hemoglobin from rabbit is 6.8. This protein will migrate toward the positive electrode during electrophoresis.

Serum Albumin – Serum albumin is the major protein found in blood plasma. This protein binds and transports a large number of smaller molecules in blood. Unlike the proteins described above, albumin is not naturally colored. However, the tracking dye bromophenol blue has been added to your serum albumin sample and some of this dye will bind and remain bound to the albumin during the electrophoretic run, turning the albumin band blue. The remainder of the bromophenol blue will migrate faster than albumin and when this free dye has migrated to the positive electrode end of the gel, the electrophoretic separation is complete. Serum albumin is a relatively acidic protein and has the lowest isoelectric point ( 4.8) of the proteins that will be used in this exercise. Thus, this protein possesses a very negative net charge at pH 8.6, and will migrate faster than hemoglobin.

5. Description of these Laboratory Exercises
In part A of this exercise, you will prepare a water-soluble extract from blue green algae and show that it contains the single major protein Phycocyanin by electrophoresis. You will also determine the charge of this protein by comparing its electrophoretic mobility to the mobilities of dyes with known charges. In part B, you prepare an alcohol extract and analyze the smaller alcohol soluble pigments by thin layer chromatography in order to identify the chlorophylls and major carotenoid pigments.
Part A. Extraction and Electrophoretic Analysis of Phycocyanin
Materials

The solutions and materials required for electrophoresis and sample handling (see Instructor Guide).
Dye Mixture 1. Bromophenol Blue, Orange G and Xylene Cyanole.
Dye Mixture 2. Cibacron Blue and Phenol Red
Glycerol Solution
Cow Hemoglobin-Albumin Mixture (Prepared as described in the Instructor Guide)

Procedure
I. Preparation of a water-soluble extract from Cyanobacteria
1. Using a water proof marking pen, place your initials on a 1.5 ml centrifuge tube and a mark 1.0 cm from the bottom of the tube.
2. Add the Cyanobactera powder until it comes up to the mark. This amount is equivalent to about 0.1 grams.
3. Add 0.75 ml of water to the tube, tightly cap the tube and shake vigorously for about 5 minutes.
4. Centrifuge the tube in a microcentrifuge for about 2 minute. The water-soluble proteins are in the top liquid layer (supernatant) while the water insoluble pigments along with cellular debris are in the pellet. If a microcentrifuge is not available, let the tube stand in a vertical position for at least 15 minutes.
5. Remove 50 ul of the top supernatant (protein extract) and add it to a tube containing 15 ul of the glycerol solution.
6. Hold this tube up to the light and note the color of the extract.

II. Electrophoresis
The procedures for the preparation of agarose gels and electrophoresis are briefly outlined below. This experiment was designed such that the samples of two students will be analyzed on one agarose gel. If the students work in pairs, four students will share one gel. The procedures described below were written for using the equipment from Modern Biology Inc. and Walter Products Inc . For other gel sizes, the procedures needs to be revised accordingly.

Preparation of 1.2 % Agarose Gels –Modern Biology Inc. Equipment

1. Place the casting tray on a level work surface and place a precleaned glass slide into the support deck. Seal both ends of the gel support deck with tape.

2. Dispense 15 ml of the electrophoresis buffer into a glass test tube and add 0.18 grams of agarose. If a balance is not available, 0.18 grams of agarose can be estimated by filling a 0.5 ml microcentrifuge tube with agarose until two-thirds full. Gently swirl the glass tube until the agarose forms a suspension.

3.Place the test tube into a boiling water bath and allow the agarose suspension to come to a vigorous boil.

4. After boiling for about 2-3 minutes, remove the tube from the bath and pour the melted agarose onto the casting deck.

5. After the gel has cooled for about 10-15 minutes, remove the tape strips and comb and transfer the gel tray to the electrophoresis chamber so that the sample wells are nearest to the black (negative) electrode.

6. Slowly fill the electrophoresis chamber with electrophoresis buffer until the gel is covered with about a 1cm layer of buffer.

Preparation of 1.2 % Agarose Gels –Walter Products Inc. Equipment

1. Place black stoppers on the ends of the casting tray and place the tray on a level work surface.

2. Dispense 25 ml of the electrophoresis buffer into a 125 or 250ml flask and add 0.30 grams of agarose. Swirl the flask until the agarose forms a suspension.

3.Place the flask into a boiling water bath or microwave and allow the agarose suspension to come to a vigorous boil. Swirl the flask at least once during the heating process.

4. After boiling for about 2-3 minutes, remove the flask from the bath or microwave and pour the melted agarose onto the casting deck.

5. Insert the comb into the comb slots on the side of the tray that contains the black strip. Use the 8 tooth side of the comb for this experiment.

6. After the gel has cooled for about 10-15 minutes, slowly remove the black end pieces and comb and transfer the gel tray to the electrophoresis chamber so that the sample wells are nearest to the black (negative) electrode.

7. Slowly fill the electrophoresis chamber with electrophoresis buffer until the gel is covered with about a 1cm layer of buffer.

II. Electrophoresis

1. Load 15 ul of each of the four samples into the sample wells as indicated below.

Sample Well
Number
1. Hemoglobin-Albumin Student Group 1
2. Protein Extract
3. Dye Mixture 1
4. Dye Mixture 2
5. Hemoglobin-Albumin Student Group 2
6. Protein Extract
7. Dye Mixture 1
8. Dye Mixture 2

2. Electrophorese at 150 volts-170 volts. During the first 6-8 minutes of electrophoresis, watch the gel closely in order to see the migration of the dyes and proteins in each sample. If necessary, remove the lid of the electrophoresis unit and note the relative position of the dyes and protein as compared to their point of application at the sample wells.

3. Resume electrophoresis for an additional 30 minutes. Remove the gels from the unit and measure the distance of each dye (in cm) and from the sample wells. Record these values below.

Data Analysis and Study Questions
1. Dye/Protein. Color Distance Migrated (cm) Relative Isoelectric Charge Point*

Dye Mixture 1( Lanes 3,7)
Bromophenol Blue Blue-Purple — 2.8
Orange G Orange —– 2.3
Xylene Cyanole Blue — 5.4

Dye Mixture 2 (Lanes 4,8)
Cibacron Blue Blue — 2.0
Phenol Red Red — 2.8

Hemoglobin (Lanes 1,5) Red –Brown – 6.8
Albumin (Lanes 1,5) Blue — 4.8

Phycocyanin ( Lanes 2,6) Blue To be determined in this experiment
*The isoelectric point is an expression of the charge on a molecule. The lower the number, the more acidic the molecule. Thus, a protein with an isoelectric point of 4.8 such as albumin is more acidic ( has a higher ratio of acidic amino acid / basic amino acid residues ) than a protein such as hemoglobin which has an isoelectric point of 6.8. The isoelectric point is defined as the -pH where a molecule does not migrate in an electric field.
_____________________________________________________________________________________________
2. Estimate the isoelectric points of phycocyanin. In order to prepare the standard curve for this operation, plot the distance migrated by the hemoglobin, albumin, bromophenol blue and xylene cyanol on the X-axis against their isoeleciric points on the Y-axis. The isoelectric points are given in the Table above.
3. The ratio of basic /acidic amino acid residues in the three proteins analyzed on your gel follows the order:

4. What is the function of Phycocyanin in Cyanobactera?

Part B. Extraction and Chromatographic Analysis of Alcohol Soluble Photosynthetic Pigments
Materials
The solutions and materials required for electrophoresis and sample handling (see Instructor Guide).
Methanol
Thin Layer Chromatography Strips
Chromatographic tubes
Small and large transfer pipets
1.5 ml microcentrifuge tubes

Procedure
I. Preparation of an Alcohol -Soluble Extract from Cyanobactera
1. Using a waterproof marking pen, place your initials on a 1.5 ml centrifuge tube and a mark 1.0 cm from the bottom of the tube.
2. Add the Cyanobactera powder until it comes up to the mark. This amount is equivalent to about 0.1 grams.
3. Add 0.5 ml of methanol to the tube, tightly cap the tube and shake vigorously for about 5 minutes.
4. Centrifuge the tube in a microcentrifuge for about 1 minute. The alcohol soluble pigments are in the top liquid layer (supernatant) while the alcohol insoluble pigments along with cellular debris are in the pellet. If a microcentrifuge is not available, let the tube stand in a vertical position for at least 15 minutes.
5. Remove 50 ul of the top supernatant (methanol extract) and add it to a fresh tube. Hold this tube up to the light and note the color of the extract.
II. Chromatography
The methanol extract contains a mixture of pigments, which you will separate by thin layer chromatography, or TLC. You will use a thin plastic strip coated with cellulose. The extract mixture will be deposited near the bottom of the strip, and the strip will then be placed vertically in methanol. As the methanol is wicked up the strip, it passes the sample and starts to carry the compounds upward with it. Different pigments dissolved in the solvent adsorb to the cellulose to different degrees. As a result, the relatively more methanol insoluble pigments remain near the bottom of the strip while other more soluble ones are carried by the methanol nearer the top.

1. Obtain one TLC strip and place in on the bench in front of you with the plastic side down.
2. Using a microliter dispenser, slowly pipette 2 ul of the methanol extract onto the center of the strip approximately 1 cm from the end of the strip. If a microliter dispenser is not available, use a small transfer pipette. For this operation, immerse the tip of the pipette into the very top of methanol extract without touching the pipette bulb. A small amount of extract will enter the tip by capillary action. Now place the tip of the pipette onto the center of the strip approximately 1 cm from the end of the strip.
3. Allow the spot to dry for at least 1 minute and then repeat the application of sample to the same spot two more times allowing the spot to dry completely between each application.
4. While you are waiting for the last extract spot to dry, obtain a plastic test tube and place it in a tube rack so that it is completely vertical.
5. Using a 3 ml transfer pipette, add 1 ml of methanol to the bottom of the tube. During this addition, attempt to avoid touching the pipette to the sides of the tube.
6. Using forceps, lower the TLC strip into the tube, sample spot end first, being careful
not to let it slide down the sides of the tube.
7. Watch the different pigments begin to separate as the solvent fronts move up the strips.
8. Remove a strip from the tube when the solvent front comes to within 1/2 cm of the top.

Data Analysis and Study Questions
1. Identify on your chromatogram the three major methanol soluble pigments. These pigments are from top to bottom: carotene (orange), chlorophyll a (green) and Xanthophylls (yellow). Xanthophylls are sometimes difficult to resolve.
2. Describe the steps that you would use to isolate carotene from Cyanobactera.
3. Describe the steps that you would use to isolate Phycocyanin from Cyanobactera.