Ion Exchange Chromatography. Teaching Kit Manual. GeNei TM. Cat No. New Cat No. KT Revision No.:

Transcription

1 Ion Exchange Chromatography Teaching Kit Manual Cat No. New Cat No. KT Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 5 Materials Provided 7 Procedure 8 Result 13 Appendix 14 Ordering Information 15 1

3 Objectives: To learn protein purification by ion exchange chromatography involving the following experiments: Purification of Lysozyme using CM-cellulose. Estimation of Lysozyme activity. Estimation of protein concentration. Principle: Ion exchange chromatography works on the basic principle that oppositely charged particles are attracted to each other. The stationary phase consists of fixed charges on a solid support. These charges can be either negative or positive. Hence, there are two types of ion exchangers i.e., cation and anion exchangers. Cation exchanger possess negatively charged groups and attract positively charged molecules, eg, Carboxymethylcellulose or CM-cellulose. Conversely, anion exchanger have positively charged groups that attract negatively charged molecules and thus separate anionic molecules. eg, Diethylaminoethyl-cellulose. Proteins are complex ampholytes i.e., they have both positive and negative charges and can be separated from a mixture of compounds on the basis of net positive or negative charge that they carry. Isoelectric point of a protein (pi) is the ph at which its net charge is zero (i.e., number of positive and negative charges are equal). Therefore, proteins will have either a net negative charge or net positive charge depending on the ph of solution and thus, it is possible to use either an anion exchanger or a cation exchanger. Bangalore Genei, Bangalore Genei,

4 In ion exchange chromatography, a solution containing protein of interest is applied to the ion exchanger. Protein binding to the ion exchanger is dependent on net charge of the protein at that particular ph and on the ionic strength of the mobile phase. Bound protein is then eluted out from the stationary phase by increasing the concentration of counter ions or by changing ph, which alters the charge on the protein. Weakly charged protein is displaced from the stationary phase with lower concentration of counter ions than highly charged protein. This results in separation of protein based upon its net charge. Extent of purification of a protein (eg., enzyme) can be determined by computing its specific activity. Specific activity is the ratio of enzyme activity to mass of protein in the sample, usually expressed as units of activity per milligram of protein (U/mg). As the enzyme is purified (through a number of steps) other proteins in the mixture are eliminated while most of the enzyme activity is retained, this results in an increase in the specific activity of the enzyme. Hence, by determining specific activity before and after purification, one can determine the fold purification and yield of the enzyme. Kit Description: Using this kit, students will carry out purification of lysozyme from chicken egg white by ion exchange chromatography. pi of lysozyme is 10.5 and it carries a net positive charge at ph below Hence, at ph 7.0 it binds to negatively charged column or a cation exchanger, CM-Cellulose supplied in the kit. Wash buffer (ph 9.0) will then be used to remove the protein that have pi less than or equal to 9.0. Following which, lysozyme will be eluted out by increasing concentration of cations. Cations compete with positively charged groups of lysozyme for binding sites on the column, resulting in the elution of lysozyme. Students will estimate lysozyme activity and protein concentration of the crude and purified samples as follows: Enzyme Activity of lysozyme is determined using the bacterium Micrococcus luteus. A suspension of intact bacteria is cloudy, absorbing light strongly at 450 nm. As lysozyme breaks down the cell walls, bacterial membranes break open due to osmotic shock, the breakdown products dissolve and the suspension becomes clearer. Thus, as lysozyme acts, the absorbance of the substrate suspension decreases. Hence, one unit of lysozyme is defined as the amount of lysozyme that will produce a decrease in absorbance at 450 nm of absorbance units/minute. 4 5

5 Protein concentration of lysozyme is estimated by measuring the UV absorbance at 260 nm and 280 nm. This value will be used to calculate specific activity and yield of lysozyme. Specific activity of lysozyme before and after purification will be estimated by comparing its activity with that of a standard lysozyme whose specific activity is known. KT40 : The kit is designed to carry out 5 lysozyme purification experiments by ion exchange chromatography. Duration of experiment: Approximately 5 hours Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival. Quantity Material KT40 Store (5 expts.) CM-cellulose 5 ml 4 C 10X Equilibration buffer (ph 7.0) 25 ml 4 C 10X Wash buffer (ph 9.0) 25 ml 4 C 5X Elution buffer 15 ml 4 C Neutralizing solution 5 ml 4 C 5M Sodium chloride 10 ml 4 C 0.5 M Phosphate buffer (ph 7.0) 25 ml 4 C Tube for mixing 1 No. 4 C Micrococcus luteus 3 Nos. 4 C Lysozyme Standard 2 x 1 mg 4 C Column 1 No. 4 C Materials Required: Equipment : Centrifuge, Spectrophotometer. Reagents : Chicken egg, Distilled water. Glassware : Beakers, Test tubes. Other Requirements: Column stand, Micropipette, Tips, Quartz cuvette. 6 7

6 Note: Read the entire procedure before starting the experiment. Handle neutralizing solution carefully as it is corrosive. Store the Micrococcus luteus (Mlu) vial at 4 C before reconstitution. Reconstitute the Micrococcus luteus vial with 0.1 ml of 0.067M phosphate buffer, ph 7.0 (PB). Store at -20 C. Use one vial as substrate for 2 experiments.gently handle the reconstituted Mlu vial. Reconstitute the lysozyme standard with 1 ml of PB. Store at 4 C and use within 3 months. Use one vial for 3 experiments. Use a Quartz cuvette to measure absorbance at A 260 and A 280. Mix CM-Cellulose column material gently to make a uniform suspension, before packing. Each packed CM-cellulose column can be used for maximum 3 times. Do not let the column go dry. Bring the reagents to room temperature before dilution. Dilute the required amount of standard lysozyme, just before use. For preparation of working concentration of reagents/ buffers, refer appendix. Procedure: Purification of Lysozyme using CM-cellulose: 1. Wash the empty column with hot water (90 C). 2. Fix the column to the stand. Remove the top cap of the column and pack the column with 2.5 ml of CM-Cellulose. 3. Remove the bottom cap and equilibrate the column with 50 ml of 1X equilibration buffer. 4. Break an egg, collect the egg white separately. 5. To 6 ml of egg white, add an equal volume of distilled water. Mix in the tube provided for 10 minutes to get a homogenous solution. 6. Adjust the ph of the egg white to 7.0, by slowly adding neutralizing solution. Note: Solution will turn slightly turbid 7. Centrifuge the egg white solution at 6000 rpm for 10 minutes. Collect the supernatant. 8. Save 0.5 ml of the supernatant for measurement of lysozyme activity, label this as crude sample. 9. Load rest of the supernatant to equilibrated CM-Cellulose column. 10. Replace the top and bottom caps of the column and incubate for 1 hour at room temperature with intermittent mixing. 11. After an hour, allow the column material to settle. Slowly pipette out or decant the supernatant without disturbing the column. 12. Wash the column with approximately 30 to 40 ml of 1X wash buffer. 13. Elute lysozyme from the column using 15 ml of 1X elution buffer. 14. Start collecting the eluate in test tubes as 2 ml fractions. Monitor OD at A 280 and pool the fractions that show A and above. Label this as eluate. 15. Wash the column with 10 ml of 1M NaCl. Replace the top and bottom caps. Store at 4 C, for next use. Note: The packed column can be reused two more times. Discard the column material after 3 uses and pack with fresh 2.5 ml CM-cellulose material. 8 9

7 Estimation of Protein Concentration: Crude sample: 1. Dilute the crude sample 20 times with PB (i.e., 0.2 ml of egg white made upto 4 ml). 2. Zero the spectrophotometer against phosphate buffer blank. 3. Measure the absorbance at A 260 and A Use the following formula to calculate concentration of protein in crude sample: Protein concentration = [(1.55 x A 280 ) (0.77 x A 260 )] x dilution factor (dilution factor = 20). = mg/ml Eluate: 1. Dilute the eluate 10 times with PB (i.e., 0.3 ml made upto 3 ml). 2. Zero the spectrophotometer against phosphate buffer blank. 3. Measure the absorbance at A Use the following formula to calculate concentration of proteins in eluate. Protein concentration A 280 x dilution factor = mg/ml = 2.55 where, dilution factor = 10 and 2.55 is the extinction coefficient of lysozyme i.e., of 1 mg/ml of lysozyme is A 280 Estimation of Lysozyme activity: 1. Dilute required amount of reconstituted standard lysozyme 1:3 i.e., (0.3 ml of standard lysozyme ml of phosphate buffer) to bring down the concentration to 0.33 mg/ml (from 1 mg/ml to 0.33 mg/ ml). Note: Store the remaining lysozyme at 4 C and use as standard for two more experiments. 2. Dilute the crude sample and eluted sample to 0.33 mg/ml based on the protein concentration estimated. For example, if the concentration is 1 mg/ml, dilute it three times with phosphate buffer to bring down the concentration to 0.33 mg/ml. 3. Zero the spectrophotometer against phosphate buffer blank. 4. Dilute required amount of Mlu in phosphate buffer such that A 450 is between 0.5 and 0.7. Note: Store the remaining reconstituted Mlu at -20 C and use as substrate for one more experiment. 5. Take 3 ml of diluted Mlu substrate in a cuvette and measure the absorbance at A 450 against phosphate buffer blank, this is A 450 at 0 second. 6. To the substrate, add 50 µl of standard lysozyme and note the time. 7. Mix the contents of cuvette for 15 seconds. 8. Measure the absorbance exactly after 60 seconds of addition of lysozyme. 9. Repeat steps 5 to 8 for the crude and eluted samples. 10. Note down the readings as in table 1. Bangalore Genei, Bangalore Genei,

8 Time A 450 Standard Crude Eluate 0 sec. 60 sec. A 450 Table 1: OD readings of lysozyme activity. Estimation of specific activity of Lysozyme: Specific activity of lysozyme in crude and eluted samples is calculated by comparing the OD readings of the samples with that of standard lysozyme, whose specific activity is known. Result: Calculate and report fold purification of the enzyme lysozyme. Crude- Sample Eluate C1 C2 C3 C4 C5 Total Total Protein Protein Specific Total Volume Conc. (in mg) Activity Yield (U) (mg/ml) C1xC2 (U/mg) C3xC4 Activity in U/mg= A 450 / min. of the test x activity of the standard * A 450 /min. of the standard Where A 450 is the difference in A 450 between 0 sec and 60 sec *Activity of standard is 48,000 U/mg. Estimation of Fold Purification: Fold purification = Specific activity of eluted sample Specific activity of crude sample This is a measure of efficiency of purification of lysozyme using ion exchange chromatography. Estimation of Yield: Yield = Protein concentration of eluate x total volume of eluate = mg 12 13

9 Appendix: Preparation of 1X equilibration buffer and 1X wash buffer: Dilute required amount of 10X buffer ten times with distilled water. (For eg., 1 ml of buffer + 9 ml of distilled water). Preparation of 1X elution buffer: Dilute required amount of 5X buffer five times with distilled water. (For eg., 1 ml of buffer + 4 ml of distilled water). Preparation of 1M sodium chloride solution: Dilute 5M sodium chloride solution five times with distilled water. Preparation of 0.067M phosphate buffer: Dilute 0.5M phosphate buffer 7.5 times with distilled water to get a final concentration of 0.067M. Ordering Information Product Size Cat # Ion Exchange 1 Pack KT40 Chromatography Teaching Kit (Consumables for 5 experiments) Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com Bangalore Genei, Bangalore Genei,

10 Notes: Bangalore Genei, Bangalore Genei, 2007