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LAB 2 PROTEIN STANDARD CURVES I. INTRODUCTION As you know, gel electrophoresis is a powerful tool for separating and visualizing biological macromolecules. You have performed agarose gel electrophoresis to separate DNA fragments in Biology 151, and most-likely in other labs as well. The next two labs we will be using a type of electrophoresis called SDS-PAGE (SDS-polyacrylamide gel electrophoresis) that is used to separate mixtures of proteins. In SDS-PAGE a detergent, sodium dodecyl sulfate (SDS), coats soluble proteins and polypeptides with a negative charge found on the molecule. SDS-coated proteins can then migrate toward the positive electrode, but at different rates depending on their relative sizes. When proteins are heated and coated with SDS they lose their three-dimensional structure, making it easier to migrate through the gel matrix. Larger 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. Heat and SDS are used to break these disulfide bridges and release the separate polypeptide units. As a result, one functional, native protein can give rise to several smaller polypeptides which may show up as distinct bands on a gel. 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.
SDS-PAGE procedures use SDS-PAGE gels that are run in vertical electrophoresis chambers. In these systems the 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 the 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.
Proteins in your samples are not visible while the gel is running, unless they are prestained with covalently attached dyes. Therefore, during a typical electrophoresis run, you should be able to see the separation of prestained the protein standards, but not the movement of any unknown proteins under study. 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. There is generally a linear relationship between the log (base 10) of the molecular weight of a protein and the distance the protein fragment migrates in the gel. Note that this linear relationship is only between the log of the molecular weight and the distance. Because of this linear relationship, we can generate an equation for the straight line that describes the log molecular weight and distance traveled. These sorts of curves are called standard curves which can be used to identify the molecular weight of unknown proteins. Of course to be able to do this, you need to know the molecular weight of the proteins, which is why we buy protein standards like those from Bio-Rad below. Table of possible pre-stained protein standards
II. PROCEDURE The procedure for today’s lab will be pretty straight forward. You will perform quantitative SDS-PAGE electrophoresis of protein standards and generate a standard curve with error bars. The key point is to develop your technique so that you will have little variation in your results. 1. Your instructor will demonstrate how to assemble the vertical gel units and install the precast SDS-PAGE gels. Be careful not to over tighten the gel clamps 2. Add electrophoresis buffer and make sure there are no leaks in the system. Fix leaks if necessary. 3. Load as many 10 ul samples of standards as you have available. This will depend on the cost of the standards and how much is available. 4. Run the gels for about 45 minutes or until the fastest moving bands get toward the ends of the gel. 5. Remove the gel from the sandwich plates. Place on light table and directly read distance traveled. Measure from the bottom of the well to the front of the band. If you have unstained standards, you will need to stain them. Staining procedure will be provided if needed, there are a couple of standard general protein stains that can be used depending on the mix of standards we have. 6. You will be provided with the molecular weights of the standards. Use these weights to generate a standard curve with error bars (refer to previous lab for procedure). Include an equation for the regression line. Remember to take the log base 10 of the molecular weights when making your standard curve.
III. MATERIALS The materials listed below are required to perform today's experiment. They are used for sample handling, and for the preparation and analysis of the SDS-PAGE gels. - Graduated cylinders and glass distilled water - precast gels - 50 ul/group of protein standards - Electophoresis buffer - Gloves, clear plastic rulers, test tube holder for hot test tubes - Microfuge tubes - white light table - Tube holders for the 0.5 ml tubes - pipettes, eppendopf and 10 ml, plastic staining trays (6" X 6") - hot blocks set to 95C for heating samples - razor blades - PC’s with SigmaPlot 11.0
IV. REFERENCES 1. Center For Advanced Training in Cell And Molecular Biology. Some of the material for today's lab came from a workshop I attended on "Separation Techniques", Dr. Frank Castora Director at Catholic University, Washington DC. 2. Davis, L.D., Dibner, M.D. and Battey, J.F. 1986. Basic methods in molecular biology. Elsevier, New York. pp 58-75. 3. Gasque, C.E. 1989. A manual of laboratory experiences in cell biology. W.C. Brown Publishers, Dubuque, Iowa. 4. Bergman, A. 1990. Laboratory investigations in cell and molecular biology. Third Edition. John Wiley and Sons, New York. 5. Hames, B.D. and Rickwood, D. 1988. Gel electrophoresis of proteins, a practical approach. IRL Press, Washington DC.
LAB 3 AND 4
MOLECULAR WEIGHT DETERMINATION OF A MIXTURE OF UNKNOWN PROTEINS I. INTRODUCTION So, based on last-week’s lab you are all familiar with SDS-PAGE and how to make a standard curve. Your task this week is to design and perform an experiment to determine the molecular weight of a mixture of unknown proteins. Your group will need to read this lab and meet with the instructor prior to this lab to present your experimental design. During these prelab meetings I typically will ask the group questions like: 1. What in simple words are you trying to do in this lab? 2. What is your hypothesis and null hypothesis? 3. What is your experimental design matrix? I will want to see it written down. 4. How are you going to analyze/quantify your data? I will want you to be specific. For this first experiment lab I was going to give the student groups a mixture of whole blood and have you characterize the plasma proteins present, but you really need to concentrate the blood proteins a bit to get good results. So instead I will provide you with the same materials as you had last week plus 50 ul of an unknown mixture of proteins. Again, your job is to design and perform an experiment to determine the molecular weight of these proteins. As the unknown protein mixture is unstained, you will need to visualize them using the coomassie staining procedure. The Coomassie dyes bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. There are a number of staining protocols out there that all involve variations on the theme of staining, then destaining the gel. Here is one that works pretty well: 1. Immerse the gel in the staining solution. 2. Microwave for 30 seconds, I have not tried this before, but it is supposed to speed it up. 3. Place the gel on a rotating platform and slowly shake until bands become visible. Takes about half an hour (microwave is used to speed up the staining process). 4. Dump off the staining solution and save for future use. 5. Rinse the gel several times with e-pure water. 6. Dump off e-pure water 7. Immerse the gel in the destaining solution. 8. Microwave for 30 seconds, again, I have not tried this before, but it makes sense that it would speed up the procedure. 9. Fold and place a several sheets of Kimwipes™ at the corner of the container (the paper has proteins and binds to the stain, speeding up the destaining process). 10. Place the gel back on a rotating platform and shake until bands become visible. This takes about half an hour. Alternatively, you can leave it overnight. 11. Destain further by replacing the paper towel, if it is necessary. The whole idea with destaining is that the coommassie stains everything, gel and bands. You have to remove most of the stain from the gel in order to be able to see the bands.
II. MATERIALS; You will be provided with the following materials: - coommasie blue staining solution, this may be made up in a kit, or be made from mix below - 50 ul/group of unknown protein mixture - 50 ul/group of protein standards - precast gels - Graduated cylinders and glass distilled water - Electophoresis buffer - Gloves, clear plastic rulers, test tube holder for hot test tubes - Microfuge tubes - white light table - Tube holders for the 0.5 ml tubes - pipettes, eppendopf and 10 ml, plastic staining trays (6" X 6") - hot blocks set to 95C for heating samples - razor blades - microwave - PC’s with SigmaPlot 11.0
Staining Solution 1 liter:
500 ml Methanol 400 ml Ultrapure water 100 ml Glacial Acetic Acid 2.5 g Coomassie Brilliant Blue R-250 Filter solution through Whatman No. 1 filter to remove impurities.
Destaining Solution 1 liter:
785 ml ultrapure water 165 ml Ethanol 50 ml Glacial Acetic Acid
III. REFERENCES: 2. Center For Advanced Training in Cell And Molecular Biology. Some of the material for today's lab came from a workshop I attended on "Separation Techniques", Dr. Frank Castora Director at Catholic University, Washington DC. 3. Davis, L.D., Dibner, M.D. and Battey, J.F. 1986. Basic methods in molecular biology. Elsevier, New York. pp 58-75. 4. Gasque, C.E. 1989. A manual of laboratory experiences in cell biology. W.C. Brown Publishers, Dubuque, Iowa. 5. Bergman, A. 1990. Laboratory investigations in cell and molecular biology. Third Edition. John Wiley and Sons, New York. 6. Hames, B.D. and Rickwood, D. 1988. Gel electrophoresis of proteins, a practical approach. IRL Press, Washington DC.
NOTES FOR FIRST EXPERIMENTAL LAB When you design your experiment, take the following into consideration. Your experiment must be completed, analyzed and an oral and written report must be completed by the following week’s lab. Lab number 4 will consist of the oral presentation of the results of your experiment to the class. Your group’s oral presentation should take the format of:
Introduction: background into what you did, why you decided to do it, what you hoped to accomplish, this may include a statement describing your null hypothesis
Methods: this should include the design of the experiment and how you physically went about performing your work
Results: this should include a presentation of your results and a discussion of their significance, it should include graphs (overheads) wherever appropriate and the statistical analysis you used to give some degree of confidence in your results
You should plan of your presentation taking about 15-20 minutes. That will allow about 10 minutes for discussion. All members should participate in the discussion and the writing up of the experiment. The group can work together in writing up a common report of their experiment. Your grade in the laboratory component of the course will be determined by the following factors: 1. Experimental design 2. Quality of your oral presentation (see rubric) 3. Quality of your lab write-up Written reports must be typed and use proper grammar. Your written reports can not just be a hard copy of your Powerpoint presentation. They should include any graphs or statistical analysis you utilized. See the first lab for a more complete format for the lab write-ups. When your group hands in its written report, it should be signed by all of the members of the group. Next to your signatures you should have a percentage (0 - 100%). This number represents the amount of time each member put into the write-up of the experiment and preparation of the oral report. The sum of these percentages must add up to 100%. Ideally each member should put in an equal amount of work. For example, if there are three people in the group, each student would ideally put in 33.3% of the work in the analysis of the data and write up of experiment and preparation of the oral report. See the first lab handout for the format to be used for the written reports.
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