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Albumin Molecule LAB 4 and 5: PROTEIN BIOCHEMISTRY Introduction
There is a fair amount of background for these two labs, so here it is. The doctrine of organic evolution is one of the most important generalizations in science. It is supported by evidence drawn from genetics, paleontology and geographical distribution and from comparative anatomy and embryology.
Each protein carries in its amino acid sequence information pertaining to its own evolutionary history and origin, and clues to the evolutionary history of the organism in which it is found. Indeed, the millions of proteins existing today are in effect living fossils.
A comparison of the amino acid sequence of the same protein in different organisms has provided a direct way to study molecular evolution. A comparison of the amino acid sequence of cytochrome C from over 80 species has revealed that the amino acid sequence of this protein from different species varies and the degree of variation corresponds to the distance that separates two species on the evolutionary tree.
That is, the greater the taxonomic difference, the more the cytochromes are likely to differ in their order of amino acid residues. For example, the cytochrome C molecules in men and chimpanzees contain 104 amino acid residues and their order of amino acids residues are exactly the same. In contrast, the cytochrome C in man differs from the cytochrome C found in yeast in 44 out of the 104 amino acid residues.
We can use the differences in the amino acids in a protein to construct a family tree of organisms that agree remarkably well with those obtained from the classical anatomical record (See Figure I). In fact, on a number of occasions, comparative protein studies have been used to clarify and expand on phylogenetic relationships that were derived from classical analysis.
Figure 1. A Family Tree Based on the Sequence of Cytochome C.
Antibodies are molecules that combine with foreign macromolecules introduced into an organism thereby rendering the foreign macromolecules inactive. The macromolecules that elicit antibody production are called antigens and are most often protein or glycoprotein in nature. Each antigen possess features that are recognized by the antibody and these features constitute the antigenic determinants or epitopes. Antibodies are frequently used to study evolutionary relationships because they recognize unique antigenic determinants along a protein molecule.
In this experiment, you will be comparing the evolutionary relatedness of the major protein in serum that is called albumin. In the first part of the experiment, you will observe the patterns of electrophoretically separated proteins present in sera from cow, goat, sheep, horse, and chicken. You will then perform an immunological procedure known as Western Blotting.
Through a series of steps, this procedure enables the investigator to visualize proteins that react with a specific antiserum after the proteins have been separated by SDS electrophoresis. Thus, the relatedness of the proteins in the serum can be compared both in terms of the number of cross-reactive proteins and in terms of the molecular weight similarities or differences of the proteins.
The first step in Western Blotting is to electrophoretically separate the serum protein samples. Next, the gel is placed against a specialized membrane made of nitrocellulose an incubated overnight. During the incubation, the proteins diffuse out of the gel and are trapped on the nitrocellulose membrane. As a result, a replica (blot) of the electrophoretically separated proteins is produced on the nitrocellulose membrane. See schematic of a blotting setup below:
Rigid Plastic ------------------------------------------------------------------------------------ Blotting Paper ------------------------------------------------------------------------------------ Gel ------------------------------------------------------------------------------------ Nitrocellulose ------------------------------------------------------------------------------------ Blotting Paper ------------------------------------------------------------------------------------ Rigid Plastic ------------------------------------------------------------------------------------
The next step is to incubate the membrane with antibodies which react with the proteins trapped on the membrane. In the present experiment, you will use antibodies that were generated in rabbits against cow serum albumin. These antibodies will bind to the membrane-trapped cow albumin and to the albumins from the other species that are structurally related to the cow albumin gelatin is added to the membrane before and during antibody incubation in order to minimize non-specific protein-antibody interaction.
Since the antigen-antibody complexes are not colored, they must be treated in some way in order to visualize them. A commonly used method involves the coupling of a color-producing enzyme to the antibody. Enzymes which catalyze the reaction of soluble, colorless substances to insoluble, colored products are often coupled to second antibodies to permit visualization.
In this experiment, the cow albumin antibody you will use has been coupled to Horseradish Peroxidase (HRP), which catalyzes the reaction below. Following incubation of the membrane with the HRP-coupled antibody, the final step in the Western Blotting procedure is to incubate the blot in a color development solution containing hydrogen peroxide and 4-chloro-1-naphthol. The immobilized HRP then coverts the 4-chloro-1-naphthol to an insoluble purple product, which is deposited at the site of the antigen bands and allows antigen visualization.
Peroxidase Chloronapthol
Soluble Insoluble Colorless Purple Substrate Product
H2O2 H2O+O2
Pre-Lab Questions
1. What is electrophoresis used for?
2. What are the levels of protein structure?
3. What is cytochrome-C and how can it be used to examine evolutionary relationships?
4. What is Albumin?
5. What is SDS-PAGE
6. What is a Western Blot?
7. What is an Antibody and Antigen
8. Make a “flow-chart” for this lab sequence (this week’s and next week’s lab)
Procedure
1. You will be working in pairs of two students for this lab where each pair sharing one gel and one blot. Your instructor will demonstrate how to assemble the vertical gel units and install the precast polyacrylaminde gels. Be careful not to over tighten the gel clamps and take care that there are no leaks in the system. Once the gel is installed and buffer has been added you can load the samples. Note that the samples should be placed in 100C hot block for 3-5 minutes prior to application. Here is a recommended loading scheme using 10ul of samples:
WELL NUMBER SAMPLE*
1 Standard Proteins 2 Cow Serum 3 Group 1 Horse Serum 4 Goat Serum 5 Sheep Serum 6 Chicken Serum _______________________________________________ 1 Standard Proteins 2 Cow Serum 3 Group 2 Donkey Serum 4 Goat Serum 5 Sheep Serum 6 Chicken Serum
2. Apply voltage until the bromophenol blue has migrated to within 1 cm of the positive electrode end of the gel. At 120 volts, this should take about 50 minutes.
3. Remove the gel from the cassette and place it in about 50 ml of transfer solution (20% Methanol) for about 10-20 minutes.
4. Wet all materials for transfer (listed below) with transfer solution. Note: gloves should be worn when handling the nitrocellulose membrane to prevent transfer of proteins from your hands to the membrane. Place one side of the gel cassette on the bench top with the inner face of the cassette facing toward you. Build “sandwich” on top of the cassette by sequentially overlaying each of the following materials onto the cassette and smoothing with a gloved index finger to eliminate air bubbles between layers:
1 One Sheet Blotting Paper 2 One Sheet Blotting Paper 3 Polyacrylamide gel 4 One Nitrocellulose Membrane 5 One Sheet Blotting Paper 6 One Sheet Blotting Paper 7 Other side of the plastic gel cassette
5. Secure the “sandwich” with several rubber bands and submerge in 60 ml transfer solution. Press down firmly on the submerged “sandwich” to squeeze out any air that may be trapped within. Incubate at room temperature for 1-2 days. If longer times are needed until the next laboratory session, place the cassettes with blots in the refrigerator in transfer solution after 1-2 days at room temperature.
This is often a good time to stop the first Week’s lab.
The second week we will stain, initiate the antibody reaction and analyze the blot, these are the steps:
Stain:
1. Disassemble the blotting “sandwich”, remove the nitrocellulose membrane blot and place it in 20 ml of distilled water. If your transfer was successful, you will see the faint blue standard proteins on your blot.
2. After 2-3 minutes, place the blot in 20 ml of protein-blot stain.
3. After 5 minutes, pour off and discard the stain, rinse the blot 3 times with 30 ml of distilled water and note the red protein bands. The major band that you should see on the regions of the blot containing the serum samples is albumin, which has a molecular weight of about 68,000. Identify this band on your blot.
4. Cover the blot with 30ml of gelatin solution (blocks non-specific sites) and reserve the remainder of the solution for antibody dilution. Incubate the blot for 30 minutes at 37o.
Antibody Reaction:
1. Dilute the antibody to cow albumin by adding 50 µl of antibody to 7 ml of fresh gelatin solution in a square petri dish. We use the same plate for the whole lab, gently swirl the plate to ensure that all surfaces of the blot are exposed to the antibody solution.
2. Place the lid on the dish, and float the dish in a water bath at 37o for 30 minutes.
Note: Great care should be taken not to bump the dishes during the incubation.
3. Decant off the antibody solution and wash for two minutes each in 40 ml of the following solutions. Manual rocking or shaking of the container should be performed during these washes.
1. TBS + NP40 2. TBS + NP40 3. TBS + NP40 4. TBS + NP40 5. TBS
Color Development Reaction:
1. While the blots are washing, the instructor should prepare the Color Development Solution by adding 7 ml of Color Development Buffer, 0.5 ml of hydrogen peroxide and 5 ml chloronapthol to 130 ml of water.
2. Pour the last wash buffer off blot and then replace with 25 ml Color Development Solution. Gently rock blot in Color Development Solution until purple bands appear. This should take about 10 minutes. Rinse the blot in water and examine both sides.
Record your results noting the number and relative migrations of proteins that are detected in each sample. Note that the bovine albumin and bovine albumin dimer in the standard protein mixture (lanes1and 5) should yield a positive reaction with the antibody. Blots may be stored protected from heat and light (between 2 sheets of black construction paper, for example).
Study Questions
1. Compare the reaction of the antibody to albumins from the various mammals. Which mammals show the greatest similarity to cow with respect to the reaction? Which show the least?
2. How does this analysis compare with the traditional taxonomic relationships reported for these animals? You may need to consult a zoology or comparative anatomy textbook to determine what is known about the relationships between cow, sheep, goat, and horse.
3. Describe how your results would have been affected if the antibody had been made in rabbits against duck albumin.
Materials:
Power Supplies Vertical electrophoresis chambers 100C hot block for microfuge tubes Eppendorf pipets and tips Gloves The following solution and materials are provided in Modern Biology 205P kit: Horse serum Goat serum Sheep serum Cow serum Donkey serum Chicken serum Antibodies to cow albumin-HRP* Protein Blot stain (Ponceau S)** Nitrocellulose (4 sheets) Blotting paper (16 sheets) 4 plastic dishes for antibody reactions Gelatin** TBS (Tris-Buffer-Saline) TBS/NP40** (Tris-Buffer-Saline + Nonidet P40) Color Development Solution** (contains Color Development Buffer, Chloronapthol and hydrogen peroxide) ***Standard proteins *This antibody was prepared by injecting rabbits with cow serum albumin. It is coupled to the enzyme horseradish peroxidase (HRP). **Prepared as described in the Instructor Guide. ***The prestained proteins in this mixture are bovine serum albumin (dimer), bovine serum albumin (monomer), ovalbumin, and myoglobin. The molecular weights of these proteins are 132,000, 68,000, 45,000, and 14,400 daltons, respectively.
References: Modern Biology 205P
Appendix I: Background on SDS-PAGE
As you know, gel electrophoresis is a powerful tool for separating and visualizing biological macromolecules. You have performed agarose gel electrophoresis to separate nucleic acids. In this experiment, you will use a type of electrophoresis called SDS-PAGE (SDS-polyacrylamide gel electrophoresis) to separate mixtures of embryo proteins.
We will use a combination of a detergent and heat to extract and denature proteins in present in zebrafish embryo. The detergent, sodium dodecyl sulfate (SDS), coats dissolved proteins and polypeptides with negative charges. The SDS-coated proteins can then migrate toward the positive electrode, but at different rates depending on their relative sizes.
When proteins are coated with SDS and heated, they lose their three-dimensional structure and take on a net negative charge. Bigger polypeptides are coated with more molecules of SDS, so the ratio of a protein’s molecular weight to its charge is approximately the same for all proteins. This means that size (molecular weight) becomes the determinant of mobility through the gel.
As you know, many large, multimeric proteins are made up of smaller protein subunits. These polypeptides are held together by a number of bonds, including bonds between sulfur atoms in the amino acids they contain. Certain treatments (heat, SDS) can be used to break these disulfide bridges and release the separate polypeptide units.
Thus, one functional, native protein can give rise to several smaller polypeptides. Myosin, for example, is a complex of 2 heavy protein chains and 4 light chains. The light chains are of two different sizes, so a purified myosin sample will form 3 separate bands when treated appropriately prior to electrophoresis.
You should be familiar with the use of agarose gels in horizontal gel chambers to perform electrophoretic separation of nucleic acids. SDS-PAGE procedures use SDS-PAGE gels that are run in vertical electrophoresis chambers. In these systems, a polyacrylamide gel is positioned in a buffer-filled chamber between two electrodes, treated protein samples are placed in wells at the top of the gel, and the electrodes are connected to a power supply that generates a voltage gradient across the gel. The negatively charged, SDS-coated proteins are then able to migrate downward through he gel toward the positive electrode. Protein size is measured in daltons, a measure of molecular weight. One dalton is defined as the mass of a hydrogen atom, which is 1.66 x 10–24 gram. Most proteins have masses on the order of thousands of daltons, so the term kilodalton (kD) is often used to describe protein molecular weight. Given that the average weight of an amino acid is 110 daltons, the number of amino acids in a protein can be approximated from its molecular weight. • Average amino acid = 110 daltons
Proteins in your samples are not visible while the gel is running, unless they are prestained with covalently attached dyes. Our samples of embryo proteins will not be stained, but the protein standards (Kaleidoscope standards) that we will also be loading into a separate well, do have dye molecules attached to their proteins. Thus during electrophoresis, we should be able to see the separation of the protein standards, but not the proteins from our embryo extracts.
As in any electrophoresis procedure, if the electric current is left on for too long, the proteins will run off the gel at the bottom. To avoid this, a blue tracking dye is mixed with the protein samples from the embryos. This blue dye is negatively charged and is also drawn toward the positive electrode. Since the dye molecules are smaller than the proteins expected in most samples, they move more quickly through the gel.
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