2) The pH of the Tris-Glycine buffer used in Part A is 8.6, what is the net charge on the protein molecules in the samples? Which electrode should each sample migrate toward during the electrophoretic separation? (4 points)

3) Explain why did Serum Albumin migrate faster than myoglobin and Hemoglobin in Part A? (1 points)

4) How would the separation of the four proteins in Part A change if the students mistakenly used the pH 9.2 buffer instead of the pH 8.6 buffer? Would the samples migrate to different electrodes? How would the migration rates change? (3 points)

5) In Part B, the pH of the Tris-Glycine buffer used is 9.2 and the pI of normal human hemoglobin (HbAHbA) is 6.9. What is the net charge on normal human hemoglobin (HbAHbA), heterozygous (HbAHbS) and sickle cell hemoglobin (HbSHbS)? Which electrode should each sample migrate toward during the electrophoretic separation? (2 points))

6) Which amino acid residue replaces glutamic acid in sickle cell hemoglobin (HbSHbS)? What is the pI and the migration rate of HbSHbS relative to HbAHbA at pH 9.2 in Part B? (2 points)

7) How would the separation of the three proteins change If Part B of the experiment was carried out at pH 8.6 instead of pH 9? Would the samples migrate to different electrodes? How would the migration rates change? (2 points

Experiment 11: Protein Agarose Gel Electrophoresis Objectives: At the end of the exercise, you should be able to 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. Undemonstrate 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 beta- hemoglobin in patients with sickle cell disease, and how protein agarose gel electrophoresis can be used in disease diagnosis. Introduction: Important background information for this experiment is provided in page 4-6. This experiment consists of two parts, Part A and Part B. Part A: Comparing Migration Directions and Rates of Four Proteins (cytochrome C, myoglobin, hemoglobin and albumin) at pH 8.6 . Part B: Comparing Migration Rates of Hemoglobin Isolated from Normal, Heterozygous and Sickle cell anemia Individuals at pH 9.2 EVERT TWO students form a group. Your group will be assigned to run either Part A or Part B. Each student will have access to the results of both experiments and therefore, will be responsible for understanding both parts. Materials on the supply bench: 1) gel running apparatus (lid matches the tank); one comb. 2) flasks for preparing agarose gel (125 ml). 3) micropippets (P20) and yellow tip boxes 4) plastic waste cup, markers and microcentrifuge tubes 5) pH 8.6 buffer for part A 6) pH 9.2 buffer for part B 7) cylinders 8) 4 protein samples for part A (on ice) 9) 3 protein samples for part B (on ice) 10) agarose powder, balance Procedure for Pouring an Agarose Gel: 1. Place gel casting tray on the top of gel support deck, the open ends of the tray should be placed next to the sides of the chamber. Insert the comb into the middle casting tray slots. Students of part B may insert the comb at the end slots. 2. Bottles and cylinders are labelled with the assigned pH values. Using 50 ml cylinder to dispense 45 ml of electrophoresis buffer (see below) into a 125 ml flask and add g of agarose to make the final concentration of 1.2% (1%: 1 gram/ 100 ml buffer). Part A: pH 8.6, 1X buffer Part B: pH 9.2, 1X buffer 3. Microwave until the suspension comes to a low boil. Make sure the agarose is completely dissolved: the suspension needs to be completely clear. Cool at room temperature for about 5 min. 4. Pour the melted agarose into the casting tray. Rinse the flask with hot water immediately. Return the flask to your tray. 5. After the gel is solidified (about 15 min), lift up the tray from the tank and rotate it by 90 degrees, and place it back on the supporting deck. 6. Slowly fill the electrophoresis chamber with the electrophoresis buffer until the gel is completely submerged. The buffer should be about 3-4 mm above the gel surface. Part A: pH 8.6, 1X buffer Part B: pH 9.2, 1X buffer 7. While the gel is submerged in the buffer gently lift the comb straight up and out of the gel. Rinse the comb immediately and return it to your tray. Return the cylinder to the assigned supply bench. 8. Assemble gel box and set the power supply to 120V. Turn on the power supply for 30 seconds to check the set-up. "rocedure for Loading Protein Samples into the Agarose Gel 1. (TA) Transfer the protein samples into separate microcentrifuge tubes. 2. Transfer protein samples from common bench to your bench. Part A Samples to be loaded into the pH 8.6 gel i. Cytochrome C: pl = 10.2 ii. Myoglobin: pl=7.2 iii. Hemoglobin (rabbit): pl=6.8 iv. Serum albumin: pl=4.8 10 ul 10 ul 10 ul 10 ul Part B Samples to be loaded into the pH 9.2 gel v. HbA HbA (normal human hemoglobin) vi. HbA HbS (heterozygous, or Trait) vii.HbS HbS (sickle cell hemoglobin) 20 ul 20 ul 20 ul 3. Carefully direct the tip of the micropipette (P20) into the top of the sample well and slowly eject the sample into the well. The sample is already mixed with 10-20% glycerol to ensure that it sediments at the bottom of the well. A tube of 1X blue loading sample is provided for you to practice loading. 1. Assemble gel box: With the power supply off, connect the cables from the tank to the power supply, red to red (positive electrode) and black to black (negative electrode). 2. Turn on the power supply and adjust the voltage to 120V. Press the Run button on the front of the power supply to start running. 3. Electrophorese until: Part A: the bromophenol blue in the serum albumin sample has migrated to within 1 cm of the positive electrode end of the gel. ABOUT 35-40 minutes. Part B: the two bands in heterozygous sample are well separated. ABOUT 30 minutes. 4. Prepare the illustration of two protein gels after electrophoresis (Part A AND B) with each lane clearly labeled with the name of the samples loaded. Cleaning Up: 1. Refill yellow tip box. 2. Return the protein microcentrifuge tubes to assigned tube racks on supply bench. 3. Dispose the tips, agarose gels into big biohazard trashcan. 4. Transfer the gel running buffer into the beaker labeled with correct pH value. 5. Clean up the tank, the comb, the gel tray with tap water. Place the tray inside the tank and make sure the lid matches the tank. Do not dry the inside of the gel tank. Experiment 11: Protein Agarose Gel Electrophoresis Objectives: At the end of the exercise, you should be able to 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. Undemonstrate 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 beta- hemoglobin in patients with sickle cell disease, and how protein agarose gel electrophoresis can be used in disease diagnosis. Introduction: Important background information for this experiment is provided in page 4-6. This experiment consists of two parts, Part A and Part B. Part A: Comparing Migration Directions and Rates of Four Proteins (cytochrome C, myoglobin, hemoglobin and albumin) at pH 8.6 . Part B: Comparing Migration Rates of Hemoglobin Isolated from Normal, Heterozygous and Sickle cell anemia Individuals at pH 9.2 EVERT TWO students form a group. Your group will be assigned to run either Part A or Part B. Each student will have access to the results of both experiments and therefore, will be responsible for understanding both parts. Materials on the supply bench: 1) gel running apparatus (lid matches the tank); one comb. 2) flasks for preparing agarose gel (125 ml). 3) micropippets (P20) and yellow tip boxes 4) plastic waste cup, markers and microcentrifuge tubes 5) pH 8.6 buffer for part A 6) pH 9.2 buffer for part B 7) cylinders 8) 4 protein samples for part A (on ice) 9) 3 protein samples for part B (on ice) 10) agarose powder, balance Procedure for Pouring an Agarose Gel: 1. Place gel casting tray on the top of gel support deck, the open ends of the tray should be placed next to the sides of the chamber. Insert the comb into the middle casting tray slots. Students of part B may insert the comb at the end slots. 2. Bottles and cylinders are labelled with the assigned pH values. Using 50 ml cylinder to dispense 45 ml of electrophoresis buffer (see below) into a 125 ml flask and add g of agarose to make the final concentration of 1.2% (1%: 1 gram/ 100 ml buffer). Part A: pH 8.6, 1X buffer Part B: pH 9.2, 1X buffer 3. Microwave until the suspension comes to a low boil. Make sure the agarose is completely dissolved: the suspension needs to be completely clear. Cool at room temperature for about 5 min. 4. Pour the melted agarose into the casting tray. Rinse the flask with hot water immediately. Return the flask to your tray. 5. After the gel is solidified (about 15 min), lift up the tray from the tank and rotate it by 90 degrees, and place it back on the supporting deck. 6. Slowly fill the electrophoresis chamber with the electrophoresis buffer until the gel is completely submerged. The buffer should be about 3-4 mm above the gel surface. Part A: pH 8.6, 1X buffer Part B: pH 9.2, 1X buffer 7. While the gel is submerged in the buffer gently lift the comb straight up and out of the gel. Rinse the comb immediately and return it to your tray. Return the cylinder to the assigned supply bench. 8. Assemble gel box and set the power supply to 120V. Turn on the power supply for 30 seconds to check the set-up. "rocedure for Loading Protein Samples into the Agarose Gel 1. (TA) Transfer the protein samples into separate microcentrifuge tubes. 2. Transfer protein samples from common bench to your bench. Part A Samples to be loaded into the pH 8.6 gel i. Cytochrome C: pl = 10.2 ii. Myoglobin: pl=7.2 iii. Hemoglobin (rabbit): pl=6.8 iv. Serum albumin: pl=4.8 10 ul 10 ul 10 ul 10 ul Part B Samples to be loaded into the pH 9.2 gel v. HbA HbA (normal human hemoglobin) vi. HbA HbS (heterozygous, or Trait) vii.HbS HbS (sickle cell hemoglobin) 20 ul 20 ul 20 ul 3. Carefully direct the tip of the micropipette (P20) into the top of the sample well and slowly eject the sample into the well. The sample is already mixed with 10-20% glycerol to ensure that it sediments at the bottom of the well. A tube of 1X blue loading sample is provided for you to practice loading. 1. Assemble gel box: With the power supply off, connect the cables from the tank to the power supply, red to red (positive electrode) and black to black (negative electrode). 2. Turn on the power supply and adjust the voltage to 120V. Press the Run button on the front of the power supply to start running. 3. Electrophorese until: Part A: the bromophenol blue in the serum albumin sample has migrated to within 1 cm of the positive electrode end of the gel. ABOUT 35-40 minutes. Part B: the two bands in heterozygous sample are well separated. ABOUT 30 minutes. 4. Prepare the illustration of two protein gels after electrophoresis (Part A AND B) with each lane clearly labeled with the name of the samples loaded. Cleaning Up: 1. Refill yellow tip box. 2. Return the protein microcentrifuge tubes to assigned tube racks on supply bench. 3. Dispose the tips, agarose gels into big biohazard trashcan. 4. Transfer the gel running buffer into the beaker labeled with correct pH value. 5. Clean up the tank, the comb, the gel tray with tap water. Place the tray inside the tank and make sure the lid matches the tank. Do not dry the inside of the gel tank