Protein Purification STRATEGY

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "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

20 Page 20 of 20

Affinity Chromatography

Affinity Chromatography PR100-02 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Affinity Chromatography Teacher s Guidebook (Cat. # BE-417) think proteins! think

More information

Classic Immunoprecipitation

Classic Immunoprecipitation 292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.

More information

PerfectFOCUS. For Preparing Low Conductivity Samples for IEF/2D-Gel Electrophoresis. (Cat. # 786-124, 786-124T)

PerfectFOCUS. For Preparing Low Conductivity Samples for IEF/2D-Gel Electrophoresis. (Cat. # 786-124, 786-124T) 357PR G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name PerfectFOCUS For Preparing Low Conductivity Samples for IEF/2D-Gel Electrophoresis

More information

Calmodulin Resin Kit

Calmodulin Resin Kit 048PR G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Calmodulin Resin Kit (Cat. # 786 552) think proteins! think G-Biosciences www.gbiosciences.com

More information

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C HIS-Select Nickel Affinity Gel Catalog Number P6611 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select Nickel Affinity Gel is an immobilized metalion affinity chromatography (IMAC)

More information

HiPer Ion Exchange Chromatography Teaching Kit

HiPer Ion Exchange Chromatography Teaching Kit HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for

More information

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı PURIFICATION AND CHARACTERIZATION OF PROTEINS

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı PURIFICATION AND CHARACTERIZATION OF PROTEINS EXPERIMENT VI PURIFICATION AND CHARACTERIZATION OF PROTEINS I- Protein isolation and dialysis In order to investigate its structure and properties a protein must be obtained in pure form. Since proteins

More information

Announcements Columbus Day 10/8 No class, 10/9 BU Monday!

Announcements Columbus Day 10/8 No class, 10/9 BU Monday! Announcements Columbus Day 10/8 No class, 10/9 BU Monday! Make up section for Tuesday discussion: Wednesday 10/10, 5-6 pm, SAR 102 Monday Section: Tuesday 10/9, 10-11 am KCB 106 Wednesday Section: Wed.

More information

6 Characterization of Casein and Bovine Serum Albumin

6 Characterization of Casein and Bovine Serum Albumin 6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function

More information

Aurum Ion Exchange Mini Kits and Columns. Instruction Manual

Aurum Ion Exchange Mini Kits and Columns. Instruction Manual Aurum Ion Exchange Mini Kits and Columns Instruction Manual Catalog # 732-6710 Aurum AEX Mini Kits, 2 pk 732-6705 Aurum AEX Mini Kits, 10 pk 732-6706 Aurum AEX Mini Columns, 25 pk 732-6707 Aurum AEX Mini

More information

Plant Genomic DNA Extraction using CTAB

Plant Genomic DNA Extraction using CTAB Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however

More information

Why would you need to purify protein?

Why would you need to purify protein? Why would you need to purify protein? Methods for Working with Protein 1. Protein Isolation A. Selection of a protein source i. tissue and cell cultures (bacteria, yeast, mammalian, etc.) ii. genetically

More information

Protocol v002 Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells

Protocol v002 Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells Page 1 of 7 Agencourt RNAdvance Cell v2 Total RNA Isolation from Cultured Cells Please refer to http://www.agencourt.com/technical for updated protocols and refer to MSDS instructions http://www.beckmancoulter.com/customersupport/msds/msds.asp

More information

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage. 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert

More information

ReadyPrep Protein Extraction Kit (Soluble/Insoluble) Instruction Manual. Catalog #163-2085

ReadyPrep Protein Extraction Kit (Soluble/Insoluble) Instruction Manual. Catalog #163-2085 ReadyPrep Protein Extraction Kit (Soluble/Insoluble) Instruction Manual Catalog #163-2085 For technical service, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723) Table

More information

LAB 11 PLASMID DNA MINIPREP

LAB 11 PLASMID DNA MINIPREP LAB 11 PLASMID DNA MINIPREP STUDENT GUIDE GOAL The objective of this lab is to perform extraction of plasmid DNA and analyze the results. OBJECTIVES After completion, the student should be able to: 1.

More information

UltraClean Soil DNA Isolation Kit

UltraClean Soil DNA Isolation Kit PAGE 1 UltraClean Soil DNA Isolation Kit Catalog # 12800-50 50 preps New improved PCR inhibitor removal solution (IRS) included Instruction Manual (New Alternative Protocol maximizes yields) Introduction

More information

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep Guide to Reverse Phase SpinColumns Chromatography for Sample Prep www.harvardapparatus.com Contents Introduction...2-3 Modes of Separation...4-6 Spin Column Efficiency...7-8 Fast Protein Analysis...9 Specifications...10

More information

Protein Purification and Analysis

Protein Purification and Analysis Protein Purification and Analysis Numbers of genes: Humans ~40,000 genes Yeast ~6000 genes Bacteria ~3000 genes Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Some proteins

More information

Genomic DNA Extraction Kit INSTRUCTION MANUAL

Genomic DNA Extraction Kit INSTRUCTION MANUAL Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview

More information

HighPure Maxi Plasmid Kit

HighPure Maxi Plasmid Kit HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer

More information

RESOURCE Q, 1 ml and 6 ml RESOURCE S, 1 ml and 6 ml

RESOURCE Q, 1 ml and 6 ml RESOURCE S, 1 ml and 6 ml GE Healthcare Life Sciences Instructions 71-7146-00 AI Ion Exchange Columns RESOURCE Q, 1 ml and 6 ml RESOURCE S, 1 ml and 6 ml Introduction RESOURCE Q and S are pre-packed columns for separating biomolecules

More information

GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps)

GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) 1 GRS Plasmid Purification Kit Transfection Grade GK73.0002 (2 MaxiPreps) (FOR RESEARCH ONLY) Sample : Expected Yield : Endotoxin: Format : Operation Time : Elution Volume : 50-400 ml of cultured bacterial

More information

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Introduction Protein extraction from tissues and cultured cells is the first step for many biochemical and analytical

More information

ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear)

ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) ReadyPrep Protein Extraction Kit (Cytoplasmic/Nuclear) Instruction Manual Catalog #163-2089 For technical service, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723) Bio-Rad

More information

Rapid GST Inclusion Body Solubilization and Renaturation Kit

Rapid GST Inclusion Body Solubilization and Renaturation Kit Product Manual Rapid GST Inclusion Body Solubilization and Renaturation Kit Catalog Number AKR-110 FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Bacteria are widely used for His

More information

Benchtop Mitochondria Isolation Protocol

Benchtop Mitochondria Isolation Protocol Benchtop Mitochondria Isolation Protocol Note: Specific protocols are available for the following products: MS850 Mitochondria Isolation Kit for Rodent Tissue MS851 Mitochondria Isolation Kit for Rodent

More information

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity

Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency

More information

ProteoMiner Protein Enrichment Kits

ProteoMiner Protein Enrichment Kits ProteoMiner Protein Enrichment Kits Instruction Manual Catalog # 163-3003 163-3006 163-3007 163-3008 163-3009 163-3010 163-3011 163-3012 For Technical Support, contact your local Bio-Rad office, or in

More information

MLX BCG Buccal Cell Genomic DNA Extraction Kit. Performance Characteristics

MLX BCG Buccal Cell Genomic DNA Extraction Kit. Performance Characteristics MLX BCG Buccal Cell Genomic DNA Extraction Kit Performance Characteristics Monolythix, Inc. 4720 Calle Carga Camarillo, CA 93012 Tel: (805) 484-8478 monolythix.com Page 2 of 9 MLX BCG Buccal Cell Genomic

More information

General western blot protocol. Guidance for running an efficient and accurate experiment

General western blot protocol. Guidance for running an efficient and accurate experiment blot protocol Guidance for running an efficient and accurate experiment Contents Introduction Solution and reagents Sample lysis Sample preparation Loading and running the gel Antibody staining Useful

More information

Nucleic Acid Quantification NUCLEIC dotmetric

Nucleic Acid Quantification NUCLEIC dotmetric PR060 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Nucleic Acid Quantification NUCLEIC dotmetric Teacher s Guidebook (Cat. # BE-318) think

More information

First Strand cdna Synthesis

First Strand cdna Synthesis 380PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name First Strand cdna Synthesis (Cat. # 786 812) think proteins! think G-Biosciences

More information

For Research Use Only Ver

For Research Use Only Ver INSTRUCTION MANUAL Quick-RNA MiniPrep Catalog Nos. R1054 & R1055 Highlights High-quality total RNA (including small RNAs) from a wide range of samples. You can opt to isolate small and large RNAs in separate

More information

Purification of membrane proteins. University)

Purification of membrane proteins. University) Purification of membrane proteins Dr.Alain Jacquet (Chulalongkorn Dr.Alain Jacquet (Chulalongkorn University) Account for about 40% of the proteins in the cell Receptors, ion channels, transmembrane transporters,

More information

CHROMATOGRAPHY Lab 3 INTRODUCTION

CHROMATOGRAPHY Lab 3 INTRODUCTION CHROMATOGRAPHY Lab 3 contributed by Mike Brundage CAUTIONS AND PITFALLS Your column is packed with Sephedex beads and is very fragile. Do not move or bump the column while running the experiment.. Be sure

More information

An In-Gel Digestion Protocol

An In-Gel Digestion Protocol An In-Gel Digestion Protocol This protocol describes the digestion of a protein present in an SDS-PAGE gel band with trypsin. The band can be taken from either a 1D or 2D electrophoresis gel. Reagents

More information

Purification of Plasmid DNA

Purification of Plasmid DNA Purification of Plasmid DNA Introduction: The growth of colonies on antibiotic medium provides phenotypic evidence that cells have been transformed. To confirm this at the genotypic level, plasmid DNA

More information

Expression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu

Expression and Purification of Recombinant Protein in bacteria and Yeast. Presented By: Puspa pandey, Mohit sachdeva & Ming yu Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.

More information

AxyPrep Blood Genomic DNA Maxiprep Kit

AxyPrep Blood Genomic DNA Maxiprep Kit AxyPrep Blood Genomic DNA Maxiprep Kit For the purification of high molecular weight genomic DNA from larger volumes of whole blood Kit contents, storage and stability Cat. No. AP-MX-BL-GDNA-10 AP-MX-BL-GDNA-25

More information

Two-Dimensional Gel Electrophoresis (2-DGE)

Two-Dimensional Gel Electrophoresis (2-DGE) - Introduction - Sample preparation - First dimension: Isoelectric focusing - Second dimension: SDS-PAGE - Detection of protein spots: staining - Imaging analysis & 2D Gel databases - Spot handling: excision,

More information

Proteomics Workflows & Technologies

Proteomics Workflows & Technologies Proteomics Workflows & Technologies Proteomics "The analysis of the entire PROTEin complement expressed by a genome, or by a cell or tissue type." Wasinger VC et al, Electrophoresis 16 (1995) Proteomics

More information

RiboZol RNA Extraction Reagents

RiboZol RNA Extraction Reagents RiboZol RNA Extraction Reagents Code Description Size N580-30ML-SAMPLE Ribozol TM RNA Extraction Reagent 30 ml N580-30ML Ribozol TM RNA Extraction Reagent 30 ml N580-100ML Ribozol TM RNA Extraction Reagent

More information

The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade plasmid DNA.

The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade plasmid DNA. INSTRUCTION MANUAL ZymoPURE Plasmid Gigaprep Kit Catalog Nos. D4204 (Patent Pending) Highlights The fastest spin-column based procedure for purifying up to 10 mg of ultra-pure endotoxin-free transfection-grade

More information

Recombinant Enterokinase Kits

Recombinant Enterokinase Kits Table of Contents About the Kits...2 Description 2 Components 2 rek Cleavage...3 Small scale optimization 3 Scale-up 4 Monitoring cleavage 4 rek Capture...5 Capture buffer considerations 5 Monitoring rek

More information

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)

More information

PowerFecal DNA Isolation Kit

PowerFecal DNA Isolation Kit PowerFecal DNA Isolation Kit Catalog No. Quantity 12830-50 50 Preps Instruction Manual Inhibitor Removal Technology (IRT) is a registered trademark of MO BIO Laboratories, Inc. and is covered by the following

More information

DNA Isolation Kit for Cells and Tissues

DNA Isolation Kit for Cells and Tissues DNA Isolation Kit for Cells and Tissues for 10 isolations of 00 mg each for tissue or 5 x 10 7 cultured cells Cat. No. 11 81 770 001 Principle Starting material Application Time required Results Benefits

More information

ZR Fungal/Bacterial DNA MiniPrep Catalog No. D6005

ZR Fungal/Bacterial DNA MiniPrep Catalog No. D6005 INSTRUCTION MANUAL ZR Fungal/Bacterial DNA MiniPrep Catalog No. D6005 Highlights Simple, efficient isolation of DNA (up to 25 µg/prep) from all types of tough-to-lyse fungi (e.g., yeast) and bacteria in

More information

ISOLATE II PCR and Gel Kit. Product Manual

ISOLATE II PCR and Gel Kit. Product Manual ISOLATE II PCR and Gel Kit Product Manual 2 Product Manual www.bioline.com/isolate PCR and Gel Kit ISOLATE II PCR and Gel Kit ISOLATE II PCR and Gel Kit 1 Kit contents 04 2 Description 04 3 Storage 04

More information

Affi-Prep Protein A Matrix Instruction Manual

Affi-Prep Protein A Matrix Instruction Manual Affi-Prep Protein A Matrix Instruction Manual Catalog Numbers 156-0005 156-0006 Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547 LIT-230 Rev B Table of Contents Section 1 Introduction...1

More information

DNA SPOOLING 1 ISOLATION OF DNA FROM ONION

DNA SPOOLING 1 ISOLATION OF DNA FROM ONION DNA SPOOLING 1 ISOLATION OF DNA FROM ONION INTRODUCTION This laboratory protocol will demonstrate several basic steps required for isolation of chromosomal DNA from cells. To extract the chromosomal DNA,

More information

Protein Precipitation Protocols

Protein Precipitation Protocols Protein Precipitation Protocols Notes: All reagents need to high purity/hplc quality. All tubes used should be new or hand cleaned thoroughly with Micro90 detergent. High quality water needs to be used

More information

Genolution Pharmaceuticals, Inc. Life Science and Molecular Diagnostic Products

Genolution Pharmaceuticals, Inc. Life Science and Molecular Diagnostic Products Genolution Pharmaceuticals, Inc. Revolution through genes, And Solution through genes. Life Science and Molecular Diagnostic Products www.genolution1.com TEL; 02-3010-8670, 8672 Geno-Serum Hepatitis B

More information

Microbiology Laboratory: MOLECULAR IDENTIFICATION OF UNKNOWN BACTERIA

Microbiology Laboratory: MOLECULAR IDENTIFICATION OF UNKNOWN BACTERIA Microbiology Laboratory: MOLECULAR IDENTIFICATION OF UNKNOWN BACTERIA Classical Microbiology courses are typically structured to introduce the identification of bacterial species using a series of biochemical

More information

TIANquick Mini Purification Kit

TIANquick Mini Purification Kit TIANquick Mini Purification Kit For purification of PCR products, 100 bp to 20 kb www.tiangen.com TIANquick Mini Purification Kit (Spin column) Cat no. DP203 Kit Contents Contents Buffer BL Buffer PB Buffer

More information

Purification of GST-tagged Proteins

Purification of GST-tagged Proteins Purification of GST-tagged Proteins User Manual Protino GST/4B Columns 1 ml Protino GST/4B Columns 5 ml January 2010 / Rev. 01 MACHEREY-NAGEL MN Table of contents 1 Components 4 1.1 Kit contents and storage

More information

Ubiquitin Interact Kit

Ubiquitin Interact Kit Ubiquitin Interact Kit Item No. 15978 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 3 Precautions 4 If You

More information

Application Guide... 2

Application Guide... 2 Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied

More information

QIAGEN Supplementary Protocol

QIAGEN Supplementary Protocol QIAGEN Supplementary Protocol Isolation of Peripheral Blood Mononuclear Cells (PBMC) and Purification of Total RNA from PBMC Using the RNeasy Micro or Mini Kit This protocol describes how to isolate PBMC

More information

AxyPrep TM Mag FragmentSelect-I Protocol

AxyPrep TM Mag FragmentSelect-I Protocol AxyPrep TM Mag FragmentSelect-I Protocol (Fragment Size Selection for Illumina Genome Analyzer and Life Technologies SoLiD) Introduction The AxyPrep Mag FragmentSelect-I purification kit utilizes a unique

More information

Environmental Water Testing: Surface Water, Groundwater, Hard Water, Wastewater, & Seawater

Environmental Water Testing: Surface Water, Groundwater, Hard Water, Wastewater, & Seawater Document: AND Sol Env 08 2013 Environmental Water Testing: Surface Water, Groundwater, Hard Water, Wastewater, & Seawater Matrix specific sample preparation and testing methods for environmental waters

More information

MagExtractor -Genome-

MagExtractor -Genome- Instruction manual MagExtractor-Genome-0810 F0981K MagExtractor -Genome- NPK-101 100 preparations Store at 4 C Contents [1] Introduction [2] Components [3] Materials required [4] Protocol 1. Purification

More information

Recommended Procedures for the Extraction of RNA. Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010

Recommended Procedures for the Extraction of RNA. Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010 Recommended Procedures for the Extraction of RNA Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010 RNA Extraction Isolates RNA from other cellular components in the

More information

UltraClean PCR Clean-Up Kit

UltraClean PCR Clean-Up Kit UltraClean PCR Clean-Up Kit Catalog No. Quantity 12500-50 50 Preps 12500-100 100 Preps 12500-250 250 Preps Instruction Manual Please recycle Version: 02212013 1 Table of Contents Introduction... 3 Protocol

More information

Dot Blot Analysis. Teacher s Guidebook. (Cat. # BE 502) think proteins! think G-Biosciences www.gbiosciences.com

Dot Blot Analysis. Teacher s Guidebook. (Cat. # BE 502) think proteins! think G-Biosciences www.gbiosciences.com PR110 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Dot Blot Analysis Teacher s Guidebook (Cat. # BE 502) think proteins! think G-Biosciences

More information

Procedure for RNA isolation from human muscle or fat

Procedure for RNA isolation from human muscle or fat Procedure for RNA isolation from human muscle or fat Reagents, all Rnase free: 20% SDS DEPC-H2O Rnase ZAP 75% EtOH Trizol Chloroform Isopropanol 0.8M NaCitrate/1.2M NaCl TE buffer, ph 7.0 1. Homogenizer-probe

More information

IMMUNOPRECIPITATION (IP) PROTOCOL

IMMUNOPRECIPITATION (IP) PROTOCOL IMMUNOPRECIPITATION (IP) PROTOCOL Immunoprecipitation is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract so that the antibody

More information

About the Kits...2 Description 2 Components 2. Factor Xa Cleavage...3 Small scale optimization 3 Scale-up 3 Monitoring cleavage 4

About the Kits...2 Description 2 Components 2. Factor Xa Cleavage...3 Small scale optimization 3 Scale-up 3 Monitoring cleavage 4 Table of Contents About the Kits...2 Description 2 Components 2 Factor Xa Cleavage...3 Small scale optimization 3 Scale-up 3 Monitoring cleavage 4 Factor Xa Capture...5 Capture buffer considerations 5

More information

Chapter 5 : Other Separation Techniques

Chapter 5 : Other Separation Techniques Chapter 5 : Other Separation Techniques The characteristics of proteins and other biomolecules that are used in the various separation procedures are solubility, ionic charge, molecular size, and binding

More information

Quantum Prep Plasmid Miniprep Kit Instruction Manual

Quantum Prep Plasmid Miniprep Kit Instruction Manual Quantum Prep Plasmid Miniprep Kit Instruction Manual Catalog #732-6100 For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-424-6723 Table of Contents Section 1 Introduction...

More information

Protein Kinase Assay Kit, Universal Cat. No

Protein Kinase Assay Kit, Universal Cat. No User Protocol 539551 Rev. 16-September-04 JSW Page 1 of Protein Kinase Assay Kit, Universal Cat. No. 539551 Introduction Protein kinases catalyze the transfer of gamma phosphate from adenosine triphosphate

More information

Pure-IP Western Blot Detection Kit

Pure-IP Western Blot Detection Kit Product Manual Pure-IP Western Blot Detection Kit Catalog Number PRB-5002 20 blots FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction The technique of immunoprecipitation (IP) is used

More information

Isolation. mircury Exosome Isolation Kit Serum and plasma

Isolation. mircury Exosome Isolation Kit Serum and plasma Isolation mircury Exosome Isolation Kit Serum and plasma Instruction manual v1.2 #300101 October 2015 Table of Contents Product summary......................................................... mircury

More information

RNAzol RT RNA Isolation Reagent

RNAzol RT RNA Isolation Reagent G e n e C o p o eia TM Expressway to Discovery RNAzol RT RNA Isolation Reagent Most effective reagent for isolation of total RNA and small RNA from samples of various origin Cat. No. E01010A User Manual

More information

Size Exclusion Chromatography

Size Exclusion Chromatography Size Exclusion Chromatography TOYOPEARL Resins for SEC TOYOPEARL TOYOPEARL TOYOPEARL HW-55 TOYOPEARL HW-65 TOYOPEARL TOSOH BIOSCIENCE TOSOH BIOSCIENCE LLC 56 Keystone Drive Montgomeryville, PA 896-967

More information

Separation by Solvent Extraction

Separation by Solvent Extraction Experiment 3 Separation by Solvent Extraction Objectives To separate a mixture consisting of a carboxylic acid and a neutral compound by using solvent extraction techniques. Introduction Frequently, organic

More information

PureLink HiPure Plasmid Filter Purification Kits

PureLink HiPure Plasmid Filter Purification Kits USER GUIDE PureLink HiPure Plasmid Filter Purification Kits For Midi and Maxi preparation of Plasmid DNA Catalog numbers K2100-14, K2100-15, K2100-16, K2100-17, K2100-26, and K2100-27 Revision Date 2 May

More information

AFFINITY HIS-TAG PURIFICATION

AFFINITY HIS-TAG PURIFICATION DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 20% ethanol. INSTRUCTIONS The resins are adapted to work mainly in native conditions

More information

Guide to Ion-Exchange Chromatography

Guide to Ion-Exchange Chromatography Guide to Ion-Exchange Chromatography Table of Contents Introduction...2-4 Protocol Samples...5 Factors Effecting Selectivity...6-7 Components of Ion Exchange Media...8-9 Experimental Conditions & Method

More information

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson,

More information

Choose your optimal tools for protein studies

Choose your optimal tools for protein studies Protein Purification Choose your optimal tools for protein studies Bacterial Baculoviral Cell free Mammalian Secreted Intracellular High yield Increased solubility Highest purity Highest yield His-tag

More information

CLONTECH. Antibodies Protocol Guide PT (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY. Innovative Tools to Accelerate Discovery

CLONTECH. Antibodies Protocol Guide PT (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY. Innovative Tools to Accelerate Discovery CLONTECH Innovative Tools to Accelerate Discovery Antibodies Protocol Guide PT3407-1 (PR99224) Published 14 September 1999 FOR RESEARCH USE ONLY Table of Contents I. Introduction 3 II. Materials Required

More information

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280.

Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. Technical Bulletin Wizard DNA Clean-Up System INSTRUCTIONS FOR USE OF PRODUCT A7280. PRINTED IN USA. Revised 4/06 AF9TB141 0406TB141 Wizard DNA Clean-Up System All technical literature is available on

More information

HiTrap Heparin HP, 1 ml and 5 ml

HiTrap Heparin HP, 1 ml and 5 ml Instructions 71-7004-00 AU HiTrap affinity columns HiTrap Heparin HP, 1 ml and 5 ml HiTrap Heparin HP is a prepacked ready to use, column for preparative affinity chromatography. The special design of

More information

RNA PowerSoil Total RNA Isolation Kit Sample (Catalog No. 12866-S) Information for Ordering Product Catalog No. Quantity 12866-25 25 Preps

RNA PowerSoil Total RNA Isolation Kit Sample (Catalog No. 12866-S) Information for Ordering Product Catalog No. Quantity 12866-25 25 Preps RNA PowerSoil Total RNA Isolation Kit Sample (Catalog No. 12866-S) Information for Ordering Product Catalog No. Quantity 12866-25 25 Preps Instruction Manual New protocol instructions: (Steps 3-5) Phenol:chloroform:isoamyl

More information

MasterPure RNA Purification Kit

MasterPure RNA Purification Kit Cat. No. MCR85102 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 114 6/2012 1 EPILIT114 Rev. A

More information

Glutathione Resin. User Manual. User Manual. Cat. Nos. 635607, 635608, 635619 PT3306-1 (071414)

Glutathione Resin. User Manual. User Manual. Cat. Nos. 635607, 635608, 635619 PT3306-1 (071414) User Manual Glutathione Resin User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Cat. Nos. 635607, 635608, 635619 PT3306-1 (071414)

More information

DP419 RNAsimple Total RNA Kit. RNAprep pure Series. DP501 mircute mirna Isolation Kit. DP438 MagGene Viral DNA / RNA Kit. DP405 TRNzol Reagent

DP419 RNAsimple Total RNA Kit. RNAprep pure Series. DP501 mircute mirna Isolation Kit. DP438 MagGene Viral DNA / RNA Kit. DP405 TRNzol Reagent Overview of TIANGEN Products DP419 RNAsimple Total RNA Kit DP430 RNAprep pure Kit(For Cell/Bacteria) DP315/DP315-R TIANamp Virus DNA/RNA Kit DP431 RNAprep pure Kit (For Tissue) Silica-membrane Technology

More information

truchip Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer

truchip Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer truchip Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer Part Number: 010144 Rev C 1 Page INTRODUCTION The truchip Low Cell Chromatin Shearing Kit with Non-ionic Shearing Buffer (PN 520084)

More information

QuickZyme Soluble Collagen Assay

QuickZyme Soluble Collagen Assay QuickZyme Soluble Collagen Assay Version June 2012 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES This package insert must be read in its entirety before using this product. Introduction Collagen

More information

U.S. Patent No. 9,051,563 and other pending patents. Ver. 1.1.3

U.S. Patent No. 9,051,563 and other pending patents. Ver. 1.1.3 INSTRUCTION MANUAL Direct-zol RNA MiniPrep Catalog Nos. R050, R05, R05, & R053 Highlights Quick, spin column purification of high-quality (DNA-free) total RNA directly from TRIzol, TRI Reagent and other

More information

Chapter 2 Antibodies. Contents. Introduction

Chapter 2 Antibodies. Contents. Introduction Chapter 2 Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining

More information

TECHNICAL BULLETIN. TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets. Catalog Number T9424 Store at room temperature.

TECHNICAL BULLETIN. TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets. Catalog Number T9424 Store at room temperature. TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets Catalog Number T9424 Store at room temperature. TECHNICAL BULLETIN Product Description TRI Reagent is a quick and convenient

More information

Aurora Forensic Sample Clean-up Protocol

Aurora Forensic Sample Clean-up Protocol Aurora Forensic Sample Clean-up Protocol 106-0008-BA-D 2015 Boreal Genomics, Inc. All rights reserved. All trademarks are property of their owners. http://www.borealgenomics.com support@borealgenomics.com

More information

Protein Stabilization Reagents. Elevated Protein Protection

Protein Stabilization Reagents. Elevated Protein Protection Protein Stabilization Reagents Elevated Protein Protection Protein Stabilization Reagents Naturally occurring proteases and phosphatases can destroy proteins you spent days isolating. Save valuable time

More information

Automation in Genomics High-throughput purification of nucleic acids from biological samples. Valentina Gualdi Operational Scientist PGP

Automation in Genomics High-throughput purification of nucleic acids from biological samples. Valentina Gualdi Operational Scientist PGP Automation in Genomics High-throughput purification of nucleic acids from biological samples Valentina Gualdi Operational Scientist PGP OVERVIEW Nucleic acid purification technologies general aspects Genomic

More information

ncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols

ncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols ncounter Gene Expression Assay Manual Total RNA and Cell Lysate Protocols v.20090807 For research use only. Not for use in diagnostic procedures. Limited License Subject to the terms and conditions of

More information

PrepTip. Reverse Phase PrepTip User Guide

PrepTip. Reverse Phase PrepTip User Guide PrepTip Reverse Phase PrepTip User Guide All text, photographs and illustrations are copyrighted by Harvard Apparatus, Inc. 2004. PrepTip is a trademark of Harvard Apparatus, Inc. Harvard Apparatus 84

More information

AxyPrep TM Mag PCR Clean-up Protocol

AxyPrep TM Mag PCR Clean-up Protocol AxyPrep TM Mag PCR Clean-up Protocol Intro The AxyPrep Mag PCR Clean-up kit utilizes a unique paramagnetic bead technology for rapid, high-throughput purification of PCR amplicons. Using this kit, PCR

More information