Protein Purification STRATEGY

Transcription

1 407PR G-Biosciences A Geno Technology, Inc. (USA) brand name Protein Purification STRATEGY For Optimizing Protein Purification think proteins! think G-Biosciences

2 INTRODUCTION... 4 ITEM(S) SUPPLIED (CAT. # )... 4 IMPORTANT INFORMATION... 5 PROTEIN ACTIVITY ASSAY... 5 PROTEASE ACTIVITY... 5 PROTEIN ESTIMATION ASSAY... 5 ELECTROPHORESIS... 5 PREPARATION OF PROTEIN EXTRACT... 5 ANALYSIS OF PROTEIN PURIFICATION... 6 PROTOCOL: PROTEIN EXTRACTION... 6 PROTOCOL: PURIFICATION... 7 I. FRACTIONATION OF PROTEIN BY LOWERING THE PH... 7 PRINCIPLE... 7 ITEM(S) REQUIRED... 7 ADDITIONAL ITEMS REQUIRED... 7 PROTOCOL... 7 BUFFER EXCHANGE & EQUILIBRATION OF THE FRACTIONS... 8 RESULTS & CONCLUSIONS... 8 II. AMMONIUM SULFATE FRACTIONATION... 9 PRINCIPLE... 9 ITEM(S) REQUIRED... 9 ADDITIONAL ITEMS REQUIRED... 9 PROTOCOL... 9 BUFFER EXCHANGE & EQUILIBRATION OF THE FRACTIONS RESULTS & CONCLUSIONS III. HYDROPHOBIC CHROMATOGRAPHY...12 PRINCIPLE ITEM(S) REQUIRED ADDITIONAL ITEMS REQUIRED PROTOCOL Page 2 of 20

3 TROUBLESHOOTING RESULTS & CONCLUSIONS IV. ION EXCHANGE CHROMATOGRAPHY (IEC)...15 PRINCIPLE ITEM(S) REQUIRED PROTOCOL TROUBLESHOOTING RESULTS & CONCLUSIONS FINAL NOTES ADDITIONAL SUPPLIES RELATED PRODUCTS Page 3 of 20

4 INTRODUCTION Protein purification of a novel or unknown protein is often a difficult process as the physical properties of the protein are unknown. Most protein purification protocols involve more than one purification method and the methods routinely use the physical properties of the protein. The Protein Purification STRATEGY kit is designed to allow researcher the opportunity to quickly establish a suitable protein purification protocol. The STRATEGY kit uses different extraction and purification techniques, allowing the researcher to determine their best protocol, without having to waste time or money on a range of purification kits. When carefully followed, protein purification strategy can be established within a few days. ITEM(S) SUPPLIED (CAT. # ) Description Size Acid Precipitation Buffer I Acid Precipitation Buffer II Ammonium Sulfate Solution Anionic Resin Column Cationic Resin Column HP Elution Buffer HP Loading Buffer Loading Buffer I Loading Buffer II Loading Buffer III Phenyl HP Column Sodium Chloride (4M) SpinOUT GT 600, 1ml 1ml 1ml 12ml 3 columns 3 columns 2 columns 10/bag Page 4 of 20

5 IMPORTANT INFORMATION Protein Activity Assay Before starting the purification of a protein, the procedures for identifying or assaying the proteins for their biological activity must be established. Protease Activity It has been widely recognized that endogenous protease activity may damage the proteins during purification procedures. Inhibition of protease activity may improve the recovery of protein to be purified. A wide spectrum protease inhibitor cocktail is generally preferred. It is highly recommended that a protease inhibitor cocktail be added to the protein extraction buffer. We supply a cocktail of protease inhibitors (ProteaseArrest, Cat. # ) for inhibiting protease activity during purification. ProteaseArrest is an excellent inhibitor of serine, cysteine and metalloproteases and therefore suitable for the protection of protein during purification. Protein Estimation Assay A reliable and reproducible method of protein estimation must be used. Several methods are available; however, a method that is free from interference from detergents, dye, reducing agents, and various other commonly used agents must be used. We recommend G Biosciences Non Interfering Protein Assay (NI Protein Assay, Cat. # ) for interference free protein assay. Electrophoresis Electrophoresis is the most widely used technique for protein analysis. The most common types of electrophoresis are (1) denaturing electrophoresis preformed after denaturing the protein sample with SDS and a reducing agent and (2) non denaturing electrophoresis. The non denaturing electrophoresis allows the protein to maintain its biological function, structure and as well as interactions with other protein (subunit species) and nucleic acid molecules. You must familiarize yourself with electrophoresis techniques. Preparation of Protein Extract The first important consideration is the preparation of protein extract from which the protein of interest is to be purified. The extraction buffer must be able to maintain the biological activity and integrity of the protein. The Extraction Buffer must also be compatible with downstream purification procedures. G Biosciences offer a wide selection of extraction buffers. Protein Extraction and Lysis Buffers (PE LB ) are a series of buffers designed for the extraction of a wide variety of protein from bacterial, yeast, animal cells and tissues. The ph of the buffers is near physiological. The PE LB buffers provide a mild environment for a wide variety of proteins from bacteria, yeast, and animal cells and tissues. Any additional agent needed can be easily added into the buffer. The use of PE LB buffer will simplify the task of Page 5 of 20

6 optimizing purification procedure and, therefore, is highly recommended. The following PE LB buffers are offered. Bacterial PE LB (Cat. # ), Yeast PE LB (Cat. # ), Mammalian Cell PE LB (Cat. # ), and Tissue PE LB (Cat. # ). For information on the PE LB series of extraction buffers, please visit our web site at Analysis of Protein Purification At each step of the purification procedure, the protein must be carefully analyzed to assess the degree of enrichment and the recovery of the protein of interest. The following routine analysis is recommended: 1. Protein concentration to monitor protein recovery 2. Gel electrophoresis for distribution characterization as well as identification of the protein in a fraction, 3. Protein activity and specific activity. PROTOCOL: PROTEIN EXTRACTION 1. Select and use an appropriate extraction buffer to extract protein from the test samples (cells and tissues). 2. Prepare 5 10ml of protein extracts to perform the entire procedure. Always use freshly prepared protein extracts. The extract should preferably contain 5 50 mg protein/ml. 3. After extraction, lysis, or homogenization, centrifuge at 30,000 x g at 4 C for minutes. 4. Collect the clear supernatant to a clean, labeled tube. This is the crude protein extract containing soluble protein. 5. The sediment is insoluble debris and may contain unbroken cells, cell organelles, and membrane bound proteins. Sediment or membrane bound protein may be further solubilized and extracted using a variety of detergents. 6. Perform Protein Purification Analysis on the Crude Extract. a. Determine the protein concentration (mg protein/ml) b. Analyze by protein electrophoresis Page 6 of 20

7 PROTOCOL: PURIFICATION Purification is a step by step procedure for removing impurities and enriching the sample with respect to the protein of interest. The following procedures must be carried out in the order recommended. After a first run, the order of purification methods may be rearranged to suit your particular needs. I. FRACTIONATION OF PROTEIN BY LOWERING THE PH Principle Proteins have the lowest solubility at their isoelectric ph (pi). When lowering the ph of the extract you would expect some protein in the extract to reach their pi and thus precipitate out of the solution giving an effective fractionation step. The precipitated protein might include the protein of your interest. The method is also referred to as acid precipitation and fractionation. Item(s) Required Description Acid Precipitation Buffer I, ph 5.0 Acid Precipitation Buffer II, ph 6.0 Size 1ml 1ml SpinOUT GT 600 Columns (Micro) 5 Additional items required Centrifuge Tubes Extraction buffers Protocol 1. Add µl Acid Precipitation Buffer I and µl Acid Precipitation Buffer II to two separate 1.5 2ml centrifuge tubes. 2. Add an equal volume of Crude Extract to each of the above tubes. 3. Invert the tube a few times and incubate for 5 minutes on ice. 4. Centrifuge at 15,000xg for 5 minutes at 4 C. 5. Collect clear lysate in clean tubes, and mark the tubes as follows: a. Label the tube AP Fraction 1, ph 5.0 b. Label the tube AP Fraction 2, ph 6.0 NOTE: It is possible that the protein of your interest might precipitate during the treatment with Acid Precipitation Buffers. In such cases, you may recover your protein from the pellets. 6. Wash the pellet one time with Extraction Buffer. Add 0.5ml Extraction Buffer to the pellet and then remove the extraction buffer after 5 10 seconds. 7. Suspend the pellet in µl of Extraction Buffer. Use a pipette tip to resuspend the pellet and allow 5 10 minutes for the pellet to dissolve. Page 7 of 20

8 8. Centrifuge the suspension at 15,000 x g for 15 minutes at 4 C and collect the clear solution. 9. Label the tube AP Fraction 3, ph Label the tube AP Fraction 4, ph 6.0 Buffer exchange & equilibration of the fractions 1. Prepare a Spin OUT column for each fraction by removing the top and then bottom caps. Place into an appropriate collection tube. 2. Mark one side of the column and ensure in all centrifugations the mark is facing outwards during centrifugation. 3. Centrifuge the column at 1,000g for 2 minutes to remove the storage buffer. This compacts the resin and removes the storage buffer. 4. Place the column in a new collection tube and remove the cap. 5. Add 0.5ml Extraction Buffer to be exchanged into to the column 6. Centrifuge the column at 1,000g for 2 minutes to remove the buffer. 7. Repeat steps 2 and 3 three more times, ensuring the buffer is discarded after each centrifugation. 8. Place the column in a new collection tube and remove the cap. 9. Slowly, apply 75µl of each fraction to the center of the SpinOUT resin. 10. Centrifuge the column at 1,000g for 2 minutes to collect the desalted protein solution. Discard the column. 11. Perform Protein Purification Analysis on the Fractions. a. Determine the protein concentration (mg protein/ml) b. Analyze by protein electrophoresis Results & Conclusions By comparing the results of acid precipitation fractionations, it would be possible to establish how your protein behaved at a particular ph and how much impurity, if any, was removed at any given ph. You may find that the protein of interest did not precipitate and only impurities (protein) were precipitated and removed from the sample, allowing a degree of enrichment. An opposite scenario is also possible e.g., the protein of interest precipitates leaving behind impurity. The precipitated protein may be later solubilized allowing a degree of enrichment. It is also possible that acid precipitation irretrievably damages the protein of your interest and in a final analysis acid precipitation may not prove a suitable procedure. Page 8 of 20

9 II. AMMONIUM SULFATE FRACTIONATION Principle In the presence of a high concentration of salts, protein precipitates out of solution. Ammonium sulfate is the most widely used salt for salting out protein or protein fractionation. Different proteins precipitate out of solution at different concentrations of salt. Thus, ammonium sulfate precipitation offers a simple and rapid method of protein fractionation. IMPORTANT: For ammonium sulfate fractionation, the crude extract or the protein solution should preferably contain < 1% non ionic detergents. Use of the PE LB series of extraction buffers is suitable for ammonium sulfate fractionation. Item(s) Required Description Ammonium Sulfate Solution (90 95%) Size 12ml Spin OUT GT 600 Columns (Micro) 5 Additional Items Required Centrifuge Tubes Extraction buffers Protocol 1. Add 0.5ml crude extract to a 2ml microfuge tube labeled Tube 1. NOTE: For best results, the crude extract should not contain any detergent. 2. Slowly, add 0.25ml Ammonium sulfate in a drop wise manner. 3. Invert the tube a few times and incubate on ice for 5 minutes. Centrifuge at 15,000x g at 4 C for 5 minutes. 4. Transfer the supernatant to a clean tube labeled Tube Centrifuge Tube 1 for 5 10 seconds and remove any residual supernatant. 6. Add 0.1ml Extraction Buffer to the precipitated pellet in Tube 1. Vortex to suspend the pellet. Mark the tube AS(~30%) Fraction 1 7. Add 0.25ml Ammonium sulfate to Tube 2. Invert the tube a few times and incubate on ice for 5 minutes. Centrifuge at 15,000xg at 4 C for 5 minutes. 8. Transfer the supernatant to a clean tube labeled Tube Centrifuge Tube 2 for 5 10 seconds and remove any residual supernatant. 10. Add 0.1ml Extraction Buffer to the precipitated pellet in Tube 2. Vortex to suspend the pellet. Mark the tube AS(~45%) Fraction Add 0.5ml Ammonium sulfate to Tube 3. Invert the tube a few times and incubate on ice for 5 minutes. Centrifuge at 15,000 xg at 4 C for 5 minutes. 12. Transfer the supernatant to a clean tube labeled Tube Centrifuge Tube 3 for 5 10 seconds and remove any residual supernatant. Page 9 of 20

10 14. Add 0.1ml Extraction Buffer to the precipitated pellet in Tube 3. Vortex to suspend the pellet. Mark the tube AS(~60 %) Fraction Add 0.5ml Ammonium sulfate to Tube 4. Invert the tube a few times and incubate on ice for 5 minutes. Centrifuge at 15,000xg at 4 C for 5 minutes. 16. Transfer the supernatant to a clean tube labeled Tube 5. Mark the tube AS(~68%) Supernatant. 17. Centrifuge Tube 4 for 5 10 seconds and remove any residual supernatant. 18. Add 0.1ml Extraction Buffer to the precipitated pellet in Tube 4. Vortex to suspend the pellet. Mark the tube AS(~68 %) Fraction Vortex all the fractions for 30 seconds and then centrifuge at 15,000x g at 4 C for 5 minutes and collect clear solutions from each fraction. Buffer exchange & equilibration of the fractions 1. Prepare a Spin OUT column for each fraction by removing the top and then bottom caps. Place into an appropriate collection tube. 2. Mark one side of the column and ensure in all centrifugations the mark is facing outwards during centrifugation. 3. Centrifuge the column at 1,000g for 2 minutes to remove the storage buffer. This compacts the resin and removes the storage buffer. 4. Place the column in a new collection tube and remove the cap. 5. Add 0.5ml Extraction Buffer to be exchanged into to the column 6. Centrifuge the column at 1,000g for 2 minutes to remove the buffer. 7. Repeat steps 2 and 3 three more times, ensuring the buffer is discarded after each centrifugation. 8. Place the column in a new collection tube and remove the cap. 9. Slowly, apply 75µl of each fraction to the center of the SpinOUT resin. 10. Centrifuge the column at 1,000g for 2 minutes to collect the desalted protein solution. Discard the column. 11. Perform Protein Purification Analysis on the Fractions. a. Determine the protein concentration (mg protein/ml) b. Analyze by protein electrophoresis c. Analyze biological activity (Optional) Results & Conclusions By comparing the results of ammonium sulfate fractionation, it is possible to determine how your protein behaved during ammonium sulfate fractionation. You may find that at a certain ammonium sulfate concentration, the protein of your interest is preferentially precipitated, allowing effective enrichment of your protein. The ammonium sulfate fractionation may be repeated by modifying the volume of ammonium sulfate added into the protein solution. Select the range of ammonium sulfate concentration at which most of your protein precipitates. Page 10 of 20

11 The acid and ammonium sulfate fractionation techniques will provide valuable information. The use of these methods may enable several fold enrichment or purification of the protein in crude extract. Depending on results either one or both methods may be used. It is preferable to first use acid precipitation, which may be immediately followed with ammonium sulfate fractionation. Page 11 of 20

12 III. HYDROPHOBIC CHROMATOGRAPHY Principle Hydrophobic Chromatography is based on the fact that protein molecules, in addition to the expected hydrophilic groups (charged groups), can have extensive hydrophobic regions. These hydrophobic regions, in media favoring hydrophobic interaction (e.g. an aqueous solution with high salt concentration) can bind to the hydrophobic ligands provided in the uncharged column matrix. Elution is brought about by decreasing the salt concentration of the eluent. In some cases, a decrease of the solvent polarity is also needed (e.g., PEG, non ionic detergents, denaturants, urea, chaotropic ions). Item(s) Required Description Phenyl HP Column HP Loading Buffer [2X] HP Elution Buffer Size 2 Columns Additional Items Required Centrifuge Tubes Extraction buffers Protocol 1. The following protocol should be carried out at room temperature, as hydrophobic interactions are weaker at lower temperatures. 2. Transfer 0.25ml crude extract or previous fraction containing the protein of interest to a 1.5ml centrifuge tube. The total protein concentration should be mg protein. 3. Add 0.25ml of [2X] HP Loading Buffer. NOTE: When working with proteins which have a tendency to precipitate in [2X] HP Loading Buffer, dilute the Loading Buffer to avoid precipitation of protein. Use the same diluted Loading Buffer for preparing the Elution Buffer, as described below. 4. Remove the bottom closure and position a Phenyl HP Column in a collection tube. 5. Dilute 5ml 2X HP Loading Buffer with 5ml Extraction Buffer 6. Equilibrate the column with 10ml diluted HP Loading Buffer. Apply [1X] HP Loading Buffer in small aliquots (2 3ml) and allow the buffer to drip and collect in the collection tube. After HP Loading Buffer is completely drained out of the column, replace the bottom closure on the column. 7. For the elution step, the protocol requires brief centrifugation of the column. Centrifugation should not be too severe as to dry the column. Centrifugation should be at such a moderate speed (~ xg for seconds) so that it Page 12 of 20

13 removes only 60 70% of the buffer from the column, leaving behind in the column 30 40% buffer. If necessary, make a trial run (before loading the protein sample) to determine an appropriate centrifugation condition. Make a note of the centrifugation condition (centrifugation speed and duration) and use the exact same condition at each step, unless specified otherwise. 8. Take five microfuge tubes and mark them as Elution Buffer 1, 2, 3, 4, & 5. Mix as follows: Tube # Diluted HP Elution Buffer (ml) HP Loading Buffer [2X] (ml) HP Elution Buffer (ml) Water (ml) 1 Elution Buffer 1 0.4ml 0.1ml 0.5ml 2 Elution Buffer 2 0.3ml 0.2ml 0.5ml 3 Elution Buffer 3 0.2ml 0.3ml 0.5ml 4 Elution Buffer 4 0.1ml 0.4ml 0.5ml 5 Elution Buffer 5 1ml 9. Apply ( ml) sample (containing mg total protein) on the column. Remove the bottom closure and allow the column to drain. Allow the column to drip until there is no buffer dripping from the column. Incubate for 5 minutes at room temperature. Collect the eluent in a collection tube and mark the tube as HP Eluent IA. 10. Position the column on a clean collection tube and centrifuge the column for a brief seconds. Mark the tube and the eluent as HP Eluent IB. 11. Elute the protein with step gradient. Starting from Elution Buffer 1, elute the protein as follows. a. Apply 0.25ml of each elution buffer (in the order listed below, one after another), incubate for 5 minutes. Spin the column as described above (at the same speed and duration), collect and mark the eluents as follows: i. Apply 0.25 ml of Elution Buffer 1 and collect the fraction and label it HP Fraction 1 ii. Apply 0.25 ml of Elution Buffer 2 and collect the fraction and label it HP Fraction 2 iii. Apply 0.25 ml of Elution Buffer 3 and collect the fraction and label it HP Fraction 3 iv. Apply 0.25 ml of Elution Buffer 4 and collect the fraction and label it HP Fraction 4 v. Apply 0.25 ml of Elution Buffer 5 and collect the fraction and label it HP Fraction Perform Protein Purification Analysis on the Fractions. a. Determine the protein concentration (mg protein/ml) b. Analyze by protein electrophoresis c. Analyze biological activity (Optional) Page 13 of 20

14 Troubleshooting If the protein of interest is detected in the fractions eluting from the column immediately after loading, in addition to the fractions eluted off the column with the elution buffer then too much protein is loaded on the column. Cut down the total protein or sample volume loaded on to the column. Results & Conclusions By comparing the results of HP Fractions, it would be possible to determine how your protein behaved during HP Chromatography. Whether the protein of interest bound to the column and at what salt concentration the protein was eluted from the column, you would be able to find the chromatographic and elution conditions for effective enrichment of your protein. If the column did not immobilize the protein of your interest, you would still be able to establish how much impurity (protein) was retained by the HP Column. Page 14 of 20

15 IV. ION EXCHANGE CHROMATOGRAPHY (IEC) Principle Ionic interaction is the basis of protein purification by the IEC. Protein contains regions of charged groups on the surface that interact with the ion exchange group immobilized on the stationary phase (column). Immobilized proteins are eluted with a salt gradient. Item(s) Required Description Anionic Resin Column, 1.5ml resin Cationic Resin Column, 1.5ml resin Loading Buffer I, 0.5M Tris, ph 6.5, 20mM NaCl Loading Buffer II, 0.5M Tris, ph 7.5, 20mM NaCl Loading Buffer III, 0.5M Tris, ph 8.5, 20mM NaCl NaCl [4M] Size 3 Columns 3 Columns Protocol NOTE: The kit is supplied with three anion and three cation chromatography columns and three sample loading buffers. Use any one column at a time and perform chromatography with any one sample loading buffer at a time. You would be able to run three chromatography runs with anion columns, using three separate Loading Buffers (I, II, & III) and three chromatography runs with cation columns, using three separate Loading Buffers (I, II, & III). In total, you would be able to run six separate chromatography runs. 1. Select one anionic column (or a cationic column) and label Column 1 and position the column in a collection tube. 2. Prepare Column Equilibration Buffer by combining 5ml Loading Buffer I with 5ml Extraction Buffer. NOTE: The Extraction Buffer should contain >10mM salt 3. Equilibrate the Column 1 with 10ml Column Equilibration Buffer. Apply 3 4ml buffer at a time and allow the buffer to drip until the column is empty of buffer. After Equilibration Buffer is completely drained out of the column, replace the bottom closure on the column. 4. For elution step the protocol requires a brief centrifugation of the column. Centrifugation should not be too severe to dry the column. Centrifugation should be at such a moderate speed (~ xg for seconds) that it removes only 60 70% of the buffer from the column, leaving behind in the column 30 40% buffer. If necessary, make a trial run (before loading the protein sample) to determine an appropriate centrifugation condition. Make a note of the centrifugation conditions, Page 15 of 20

16 centrifugation speed and duration, and use the same condition at each step, unless specified otherwise. 5. Mix Loading Buffer 1 with increasing amounts of 4M NaCl, as follows: Tube # Elution Buffer # Loading Buffer I (µl) NaCl [4M] (µl) Final NaCl (mm) 1 Elution Buffer Elution Buffer Elution Buffer Elution Buffer Elution Buffer Elution Buffer Elution Buffer For the best result, use the crude extract that has been subjected to ammonium sulfate fractionation, as described above. The samples must be first dialyzed 3 4 hours in extraction buffer (containing >20mM NaCl) before running IECchromatography. Mix the appropriate sample with the Loading Buffer 1 as follows. 7. Mix 0.25ml sample with 0.25ml Loading buffer I [0.5M Tris, ph 6.5, 20mM NaCl]. 8. Apply the sample ( ml) (containing 0.4.6mg total protein) to the column. Incubate for 5 minutes. 9. Remove the bottom closure and allow the column to drain. Allow the column to drip until there is no buffer dripping from the column. Collect the eluent in a collection tube and mark the tube as IE Eluent IA. 10. Position the column on a clean collection tube and centrifuge the column for a brief seconds. Mark the tube and the eluent as IE Eluent IB. 11. Elute the protein from the column by applying 0.25ml of each of the following elution buffers, one after another in the following order and collect eluent, as follows: a. Apply 0.25 ml of Elution Buffer 1 and collect the fraction and label it IEC Fraction 1 b. Apply 0.25 ml of Elution Buffer 2 and collect the fraction and label it IEC Fraction 2 c. Apply 0.25 ml of Elution Buffer 3 and collect the fraction and label it IEC Fraction 3 d. Apply 0.25 ml of Elution Buffer 4 and collect the fraction and label it IEC Fraction 4 e. Apply 0.25 ml of Elution Buffer 5 and collect the fraction and label it IEC Fraction 5 Page 16 of 20

17 f. Apply 0.25 ml of Elution Buffer 6 and collect the fraction and label it IEC Fraction 6 g. Apply 0.25 ml of Elution Buffer 7 and collect the fraction and label it IEC Fraction Perform Protein Purification Analysis on the Fractions. a. Determine the protein concentration (mg protein/ml) b. Analyze by protein electrophoresis c. Analyze biological activity (Optional) 13. Repeat the procedure described above with the remaining columns (anionic and cationic) using the Loading Buffer II and III, respectively. Use the same scheme for preparing the Equilibration Buffer and Elution Buffer. Troubleshooting If the protein of interest is detected in the fractions eluting from the column immediately after loading, in addition to the fractions eluted off the column with the elution buffer then too much protein is loaded on the column. Cut down the total protein or sample volume loaded on to the column. Results & Conclusions By comparing the results of IEC Fractions, with (anionic or cationic) columns and under different loading and elution buffer conditions it would be possible to determine how your protein behaved during IEC chromatography. You would be able to find chromatographic loading and elution conditions for obtaining effective enrichment of your protein. Page 17 of 20

18 FINAL NOTES The fractionation techniques outlined above will provide researchers information necessary for developing a strategy for protein purification. 1. Acid fractionation and/or ammonium sulfate fractionation should be used first to fractionate the crude protein extract. If Acid Precipitation Fractionation can be used for fractionation of the protein of your interest, it should be used first followed by ammonium sulfate fractionation. NOTE: High speed ultra centrifugation may also be used for initial fractionation of protein in crude extract, e.g., when protein solution or protein extract is centrifuged at ultra high g force, the centrifugal force allows sedimentation of high mol. wt. protein molecules, cellular fractions, and cell organelles. It normally requires prolonged centrifugation in refrigerated ultra centrifuges. Ultra centrifugation may, therefore, be used for the separation of high mol. wt. protein from the low mol. wt. protein molecules. Ultra centrifugation is generally suitable for separation of >1000kd molecules from low molecular molecules. 2. After acid and/or ammonium sulfate fractionation, follow hydrophobic and/or ion exchange chromatography. When both chromatography are used, one after another, it will provide a fairly high degree of protein purification. NOTE: Gel filtration or permeation chromatography may also be used for the separation of high mol. wt. protein from the low mol. wt. protein molecules. Following acid and/or ammonium sulfate fractionation and prior to running the hydrophobic and/or ion exchange chromatography, gel filtration chromatography may be introduced to separate the high mol wt. protein from the low mol. wt. protein molecules. Collect the active fractions and then perform HP and IEC chromatography. 3. For even higher purification, additional affinity based chromatography, gradient ultra centrifugation, iso electric, chromo focusing, or electro elution techniques may be employed. ADDITIONAL SUPPLIES After optimizing the protein purification strategy, the reagents, columns, and supplies provided in the kit may be ordered separately. In addition, custom made columns and buffers can also be obtained for further purification works. Page 18 of 20

19 RELATED PRODUCTS Download our Protein Purification Handbook. protein purification handbook/ For other related products, visit our website at or contact us. Last saved: 7/24/2012 CMH Page 19 of 20

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