Lab3-Protein Purification

.pdf

School

Arizona State University *

*We aren’t endorsed by this school

Course

181

Subject

Chemistry

Date

May 24, 2024

Type

pdf

Pages

Uploaded by BrigadierRockSnail36

Lab 3-1 LAB 3 PEROXIDASE EXTRACTION AND PURIFICATION FROM HORSERADISH ( Armoracia rusticana ) ROOTS Name:_____________________________________________ Date_____________________ Dear Student: If you’re typing directly into this document, please use BLUE font color for anything you type so I can tell it apart from what I have written. INTRODUCTION Protein purification from biological tissue is a critically important skill that any lab biologist should be familiar with. In this lab exercise, we will learn about how biologists purify proteins for future use. Among the future uses could be chemical analysis, medical uses and others. We will try to purify a specific protein called peroxidase. The enzyme we use in this lab will be used by us in lab 3 and we will also investigate this enzyme in our IRPs, so this is a very important enzyme to learn about. We will purify this protein from roots of horseradish. The structure of this enzyme is shown below as both a surface model and a ribbon model. As you can see, there are many alpha helix structures in this protein. In addition, the active site of the enzyme is dominated by an iron heme group. The protein is slightly negative at cellular pH. Plant peroxidases chemically reduce harmful H 2 O 2 and other similar reactive molecules to water by oxidizing some organic substrate. H 2 O 2 and other similar reactive molecules are very harmful to biological tissues because they oxidize important biological molecules (they cause oxidative stress). So peroxidase is an important stress enzyme that plants use to cope with the oxidative stress caused by H 2 O 2. Without this enzyme, plants (and even animals) would have an abnormal buildup of H 2 O 2 , which could lead to some damaging results for the cells. One way to see this reaction is to react H 2 O 2 with a synthetic chemical called guaiacol. The product, called tetraguaiacol is an amber color and can easily be detected using a spectrophotometer:

Lab 3-2 Knowing the reaction above will be very useful toward understanding complete the procedures of this lab. The goal of this lab is to familiarize you with the techniques of protein purification and analysis. This lab will be conducted over a three-week period. First, we will start with a crude protein extraction and purification to separate proteins from other macromolecules. In the second lab meeting, we will specifically purify peroxidase from other proteins. Finally, in the third lab, we’ll analyze how well we did. 3A. CRUDE TOTAL PROTEIN EXTRACTION AND PARTIAL PURIFICATION BY SALTING OUT In 3A, you will extract and partially purify all proteins from root tissue of horseradish plant. The goal is to extract proteins from the cells and tissues of the root and to separate proteins from other macromolecules, such as carbohydrates and nucleic acids. With most proteins, purifications must be done under cold conditions to prevent denaturation. The advantage of working with peroxidase is that it is heat-stable, so we can work at room temperature for long periods, but should be stored in a frozen state. PROCEDURE Reagents and Materials: 1. Horseradish roots 2. Homogenization buffer  0.1 M phosphate buffer, pH 7.0. This buffer helps control the pH 3. 25 mM phosphate buffer, pH 7.5 4. Ammonium sulfate  salt used to precipitate proteins 5. Cheesecloth  used to filter homogenized chicken to remove large unhomogenized chunks and to remove lipids 6. 50 ml centrifuge tubes pre chilled 7. Blender  for homogenization 8. Desalting column 9. Microfuge tubes 10. Disposable pipets 11. Pipet tips 12. Weigh boats 13. 100 ml beaker (2/grp) pre chilled 14. 250 ml beaker (2/grp) pre chilled 15. 125 ml e flask (1/grp) pre chilled 16. Small petri dish (1/grp) pre chilled 17. Stir plates (1/grp) 18. Stir bars (1/grp) 19. Ice 20. Sharpies 21. 50 ml graduated cylinder (1/grp) 22. Ultracentrifuge at 5 o C 23. Ring stand with clamps (1/grp)

Lab 3-3 Homogenization, Filtration and Centrifugation 1. WEAR GLOVES THROUGHOUT THE WHOLE PROCEDURE. Horseradish can be irritating to skin and some of the chemicals we use may be skin and eye irritants. 2. Tissue Preparation — Obtain about 50 g horseradish root and peel/discard the skin 3. Chop up the root into small pieces with a knife. Record the actual weight of the chopped up you used after removal of extraneous tissues: _______g 4. Soluble Protein Extraction — Place the minced tissue and 75 ml of cold 0.1 M phosphate buffer in a blender and put the top on the blender. Homogenize the tissue 4x in 30 second bursts (or maybe more-ask your instructor). 5. Filtration — Obtain 4 layers of cheesecloth and place them over a 250 ml beaker. Pour homogenization buffer through the cheesecloth to pre-wet it and then squeeze out the excess buffer from the cheesecloth. Discard the buffer in the beaker down the sink. Now pour the homogenized tissue onto the cheesecloth in two or three batches. In between batches, push the chunks to extract as much liquid as possible through the cheesecloth using a spatula or the bulb end of a transfer pipette. Squeeze out the cheesecloth to get all remaining filtrate through the cheesecloth. Once all the homogenate has been filtered, you are ready for the next step. The cheesecloth will remove unhomogenized chunks of tissue as well as lipid from the solution. 6. Centrifugation — Put 25 ml of the filtered sample into a pre-chilled 50 ml centrifuge tube (a clear round bottom tube with a screw-on cap). Label the tube by writing on it with a sharpie pen (don’t use label tape). Balance the tubes following the directions of your lab instructor. Centrifuge the balanced tubes (opposite each other in the centrifuge) at 3000 x g for 20 min. 7. Save two 1 ml aliquots of the supernatant (aka crude homogenate) into microfuge tubes and label as “C. H., Grp #, & Lab section”. Keep these samples in an ice bath at your station until the end of lab. The instructor will freeze these at the end of class for later analysis 8. Measure and record the volume of the remaining supernatant in a graduated cylinder. Volume of the supernatant ____________ ml 9. Discard the pellet that is at the bottom of your centrifuge tube. Salting-out Proteins 10. Ammonium Sulfate Precipitation — Ammonium sulfate is a salt that, when added to a protein solution, will cause the proteins to come out of solution by disrupting the hydration sphere of the

Lab 3-4 dissolved proteins. This allows us to separate proteins from carbohydrates and nucleic acids, which stay in solution. Doing so will also let us concentrate the proteins by first precipitating them out of a large volume and then later re-dissolving them in a smaller volume. To do this, we will add solid (NH 4 ) 2 SO 4 to our centrifuged supernatant slowly until we reach a salt saturation level of 80% (0.57 g (NH 4 ) 2 SO 4 per ml of filtrate). a. Knowing the volume of the supernatant, and knowing that you need 0.57 g of (NH 4 ) 2 SO 4 per ml of filtrate, obtain the total amount of (NH 4 ) 2 SO 4 that you’ll need and write down this amount: Amt. of (NH 4 ) 2 SO 4 needed: ________________________g b. Slowly (over a period of 15 min) add 0.57 grams of ammonium sulfate per 1 ml of your centrifuged protein solution. It is best to perform this step in a chilled beaker on a magnetic stirrer. Place your sample into a beaker. Use a small magnetic stir bar to keep things mixed up. c. Avoid stirring too violently because this could shear (tear up) the proteins. If you see too many large bubbles forming then you are shearing your proteins. d. Stir for an additional 15 min after adding the ammonium sulfate to give the salt a chance to dissolve. 11. Gently pour the salted-out solution into a clean centrifuge tube. Avoid pouring out the stir bar and also avoid the undissolved salt crystals that may be at the bottom of the beaker. 12. Centrifugation — Centrifuge the sample as before in a pre-chilled balanced centrifuge. At the end, gently pipet the supernatant into a separate 50-ml blue-capped sample tube labeled “SUPER” + GRP# and section…etc. Give this tube to your instructor for freezing. 13. Keep the pellet, consisting of precipitated proteins, in the centrifuge tube. QUESTIONS 1. Explain the purpose of why we salted out the proteins. Why was it done and how does it work? 2. Why do we need to remove the (NH 4 ) 2 SO 4 salt before we go on to future steps? Briefly explain how the salt is removed from the protein.

Lab 3-5 3B. PEROXIDASE PURIFICATION USING GEL FILTRATION AND ION EXCHANGE CHROMATOGRAPHY Next, we will use ion exchange chromatography to separate proteins based on their charge. However, we must first remove the ammonium, sulfate salt that was used during the last lab period. Desalting the Protein Sample We must remove the ammonium sulfate salt from the protein pellet. High concentrations of salt, such as ammonium sulfate, can interfere with subsequent protein purification steps so removing this salt is necessary. To remove the salt, we will use a chromatography technique called gel filtration chromatography. Gel filtration chromatography separates different chemicals by their different sizes. The process of gel filtration chromatography employs a column (see figure below) that contains a buffer called the mobile phase and a semi solid (usually) material called the stationary phase. The sample to be desalted is placed in the column sample reservoir and gravity is used to pull the sample through the first frit (or filter) and into the column. Since the stationary phase consists of beads with small pores in them, the salt, which is a small molecule compared to the proteins, enters the pores and therefore spends more time in the beads. The larger proteins do not pass through the pores and simply go around the beads. Therefore, the larger proteins move faster through the column than the smaller salt ions. At the bottom of the column, the proteins come out (elute) first followed by the salt. This results in separation of the proteins from the salt. Once the sample has been desalted, the proteins can now be subjected to another kind of chromatography called ion exchange chromatography, which we’ll begin on the second day of the procedure.

Lab 3-6 Procedure for Gel Filtration Chromatography 1. Designate a small container for buffer waste and label it. Also, get a large plastic container and fill it with ice to use as ice storage. 2. Resuspend ammonium sulfate pellet — add 2 ml of 25 mM phosphate buffer to the ammonium sulfate pellet. Gently mix the buffer and the solid material until the pellet dissolves. Keep on ice as much as possible during the procedure. 3. Be sure that no buffer remains above the top frit of the column. If there’s buffer above the top frit, then use a disposable transfer pipet to remove it and discard into your waste container. 4. Load the desalting column — load 3 ml of the mixture on the desalting column. Allow the liquid to drain to the frit (the plastic cover on the column resin). The column will only drain if there is pressure by fluid above the frit. When you load your sample into the sample reservoir this creates pressure on the fluid (buffer) inside the column. The result is that the buffer elutes out of the column. Discard the flow through, which is mostly buffer. 5. Elute the sample from the desalting column — add 5 ml 25 mM phosphate buffer to the column and collect the flow through in a 15 ml blue-capped tubed labeled “Desalted” as well as your group number and lab section. The flow through contains the peroxidase and all other proteins minus the salt. 6. Remove the desalting column from the ring stand and record the volume of the flow-through in the tube ____________ ml 7. Save two 1 ml aliquots of the desalted material in two microfuge tubes and label as desalted fraction with your group number and your lab day (two labels better than one: one label on side and one on cap). Keep these as well as the larger 15ml tube on ice. Now that the ammonium sulfate has been mostly removed, we can now use ion exchange chromatography to separate proteins based on their charge. Different proteins have different total charges at cellular pH. Some are weakly or strongly negative (anions) and others are weakly or strongly positive (cations). Protein total charge comes from the types and number of charged amino acids they contain. (Remember, some amino acids are negative while others are positive. If a protein has lots of negative amino acids on its surface, then that protein will be strongly negative.)

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version