Experiment 11. Protein Agarose Gel Electrophoresis. VY NGUYEN 8 April 2016

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1 Experiment 11 Protein Agarose Gel Electrophoresis VY NGUYEN 8 April 2016 ABSTRACT 1. Understand the concepts of protein structures, isoelectric point of amino acids and proteins. 2. Understand the principle of separation in protein agarose gel electrophoresis. 3. Demonstrate laboratory techniques used in running a protein agarose gel (preparing gels, loading samples with micropippets, and electrophoresis). 4. Understand the molecular difference between normal and mutant forms of betahemoglobin in patients with sickle cell disease, and how protein agarose gel electrophoresis can be used in disease diagnosis.

2 Experiment 11: Protein Agarose Gel Electrophoresis Vy Nguyen 1 1. ( 2 points) What is the basis of separation of the four proteins in Part A of the experiment? Relate your answer to the pi of these proteins. Separation of Protein using "Native" or "nondenaturing" gel electrophoresis is based on net charge, which is dependent on the relative relationship between its isoelectric point (pi) and the ph of the gel running system. When ph is less than the pi, the amino acid is protonated and has a positive charge. When ph is greater than the pi, the amino acid is deprotonated and has a negative charge. The ph of the gel in this experiment is 8.6, and the pi, which is determined by the ionizable basic and acidic side chains of the constituent amino acids, are as follow: Cytochrome (10.2), Myoglobin (7.2), Hemoglobin (6.8), Serum Albumin (4.8). 2. (2 points) How would the separation of the four proteins in Part A of the experiment change if the students mistakenly used the ph 9.2 buffer instead of the ph 8.6 buffer? Explain. If the students mistakenly used the ph 9.2 buffer instead of the ph 8.6 buffer, the charge (positive/negative) would have remain the unchanged, but the strength of the charge would change. For example, Cytochrome C still has a positive charge because its pi (10.2) is still greater than its ph (9.2). However, because ( = 1) is less than ( = 1.6), Cytochrome C would not be as positive. Based on the same logic, myoglobin, hemoglobin, and serum albumin would be more negative because of the increase in ph. 3. (2 points) What is the basis of separation of the HbA and HbS hemoglobin in Part B of the experiment? Relate your answer to the mutation found in HbS. The separation of the HbA and HbS hemoglobin in Part B of the experiment depends on the relationship between the pi points and the ph. The HbA has a glutamine amino acid where the mutant form has a valine. This means that HbA is more acidic and has a lower pi than the mutant form, which is not as acidic. The mutant will travel a greater distance towards the cathode because of its higher pi. 4. Define the four levels of protein structures. ( 2 points) Primary structure: Amino acid sequence Secondary structure alpha helices and beta sheets Tertiary structure 3 D shape from interactions between R groups of various amino acids Quaternary structure Only for proteins composed of more than one polypeptide chains 5. a) The pi of HbA is about 6.8. Do you expect the pi of HbS to be less than 6.8 or greater than 6.8 based on the fact that HbA carries two more negatively charged amino acids per molecule than HbS? (2 points) The pi of HbS would be greater than 6.8 because HbS has fewer negatively charged particles. b) If Part B of the experiment was carried out at ph 8.6 instead of ph 9.2, do you expect to see similar separation of the two forms of hemoglobin? Why or Why not? (2 points) The pi of normal and mutant hemoglobins are both less than the gel at ph 9.2 and ph 8.6 therefore they would still have a net negative charge, and thus we would expect to see similar separation of the two forms of hemoglobin. Because the ph is lower, the separation of both forms of hemoglobin would increase.

3 Experiment 11: Protein Agarose Gel Electrophoresis Vy Nguyen 2 6. ( True or False) The net electric charge carried by a protein varies at different phs of the environment. (1 points) True 7. Use your biology textbook as a guide, hand draw sample structures of the following types of amino acids in pencil: (2 pts) (DO NOT copy and paste image files) i. one acidic amino acid ii. one basic amino acid iii. one non polar amino acid iv. one polar, uncharged amino acid 8. ( 4 points) Present the illustration of two protein gels after electrophoresis (Part A AND B) with each lane clearly labeled with the name of the samples loaded (wells, anode and cathode).

4 Experiment 11: Protein Agarose Gel Electrophoresis Vy Nguyen 3 1. Understand the subunits of proteins: amino acids Be able to label amino group, carboxyl group and side group Provided with the structure of amino acids, be able to classify them into non polar, polar or charged amino acids i. Non polar structures ones with a phenyl group attached or a carbon chain at the end. ii. polar structures have an hydroxide, thiol, or primary amine group with a ketone at the end iii. Worry mainly about basic and acidic iv. acidic structures have a carboxylic acid

5 Experiment 11: Protein Agarose Gel Electrophoresis Vy Nguyen 4 v. basic structures has a primary or secondary amine with a charge. NO KETONE on R group Know which two amino acids are acidic amino acids, which three amino acids are basic amino acids, under what condition? i. Acidic amino acids are the following under neutral ph 1. aspartic acid 2. glutimic acid ii. Basic amino acids are the following under neutral ph 1. lysine 2. Arginine 3. Histidine Any amino acid can be charged under appropriate condition. True or False? True 2. Define the four levels of protein structures. a. Primary the basic polypeptide chain of amino acids. Linear fashion b. Secondary includes helices and beta sheets c. Tertiary The alpha helices and beta sheets react with each other making a 3d structure d. Quaternary multiple tertiary proteins come together (2 or more)

6 Experiment 11: Protein Agarose Gel Electrophoresis Vy Nguyen 5 3. Understand the concept of pi The definition the point at which the protein does not migrate in an electric field (isoelectric point) Know the relationship between acidity of amino acid or proteins and pi If the amino acid is positively charged > acidic (lower pi) If the amino acid is negatively charged > basic (higher pi) Be able to determine the charge of an amino acid or a protein given its pi and environmental ph ; then predict its migration direction in an agarose gel if the pi is larger than the ph then the amino acid is positively charged. It will move toward the cathode. if the pi is smaller than ph then the amino acid is negatively charged. It will move toward the anode. 4. Understand the difference between normal hemoglobin and mutated hemoglobin in sickle cell patients; be able to compare the migration rates of the two types of hemoglobin (HbA and HbS) based on the nature of the mutation. In hemoglobin S (sickle cell anemia) a single glutamic acid residue on the Beta chain is replaced by valine. This causes the HBB sequence to go from HbA to HbS. This causes the net charge to change and the shape of the protein. The Hemoglobin S is deoxygenated. The hemoglobin molecules are negative charge and move toward the anode when the buffer ph is at 9.2. The isoelectric point of normal hemoglobin is 6.9, but hemoglobin S has a distinct two fewer negative charges. This means that it would move less distance to the anode compared to normal. 5. Understand the separation principle of protein agarose gels (native or non reducing gel electrophoresis) What determines the direction of movement of the proteins? the migration of the protein through an agarose gel is dependent on the net electric charge on the protein, intensity of the electric field, and the ph of the ionic strength of the buffer What determines the rate of movement of the proteins? the rate of movement of the proteins is determined by the difference between the isoelectric point and the ph of the buffer. Does the molecular weight of a protein affect its migration? no Does the separation requires proteins to be denatured? no Does the protein change its shape while it is moving in the gel? no 6. Properly load a sample into an agarose gel. Assemble the gel apparatus and connect to the power supply correctly (the wells was set at the end of the gel). ok

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