His60 Ni Superflow Cartridges User Manual
Contents
1. 15 His60 Ni Superflow Cartridges User Manual VI Troubleshooting Guide Table 3 Troubleshooting Guide for His60 Ni Superflow Cartridges Description of Problem Possible Explanation Optimize bacterial expression Poor expression of target protein si conditions e Decrease temperature to 25 C or lower during induction to minimize inclusion body formation Target protein forms inclusion bodies Solubilize inclusion bodies and perform the purification in the presence of 8 M urea or 6 M guanidinium HCl Inefficient target extraction Use our xTractor Buffer Inaccessible polyhistidine tag A a M urea or Low target yield Increase wash volume or add intermediate wash at 60 mM imidazole This can result in partial loss of target protein Clogged column Apply only clarified extract and decrease the amount of loaded sample Low flow rate Vi al Treat sample with Benzonase or cea sal DNase I as described in Section V A Use low UV absorbance imidazole in the buffers Impurities in eluate Insufficient washing Can not detect target protein by UV Perform a Bradford protein assay on collected fractions to identify target protein in eluate Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third pa2. 635671 e 500 ml Cat No 635625 ProteoGuard EDTA Free Protease Inhibitor Cocktail Cat No 635673 Complete easy to use protease inhibitor cocktail that is EDTA free can be used on IMAC resins without interfering with protein binding e 10x 100 ul Cat No 635673 Antibodies for detection of tagged proteins e 6xHis mAb HRP conjugate albumin free 100 ul Cat No 631210 e 6xHis Monoclonal Antibody albumin free 200 ug Cat No 631212 e 6xHN Polyclonal Antibody 200 ul Cat No 631213 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 9 of 15 His60 Ni Superflow Cartridges User Manual V Sample Preparation amp Purification PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING Use this procedure to a prepare your his tagged protein sample for b automated or c manual purification using His60 Ni Superflow Cartridges PT5143 1 030212 PROTOCOL Extracting Proteins from Cells This procedure has been optimized for extraction of proteins from fresh or frozen cell pellets using xTractor Buffer The extraction volumes can be adjusted as long as 20 ml of x Tractor Buffer are used per 1 g of cell Resuspend the cell pellet Add 20 ml of xTractor Buffer to 1 g of cell pellet Mix gently Pipet the mixture up and down to fully resuspend the pellet Optional step lysozyme amp DNase I protease inhibitor Add 40 ul of 1 unit ul DNase I solution and 200 ul of 100X lysozyme solution Add E
3. Cap His60 Ni Superflow Cartridges 5 x 5 ml Cat No 635679 e 5 His60 Ni Superflow Cartridges 5 ml each e 5 Top Caps e 5 Snap Off End Caps His60 Ni Superflow Cartridge Purification Kit 5 x 5 ml Cat No 635678 e His60 Ni Superflow Cartridges 5 x 5 ml Cat No 635679 e 1 His60 Ni Buffer Set Cat No 635665 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 6 of 15 His60 Ni Superflow Cartridges User Manual lll Additional Materials Required A Equipment For His60 Ni Superflow Cartridges you will need the following equipment e A suitable liquid chromatography system LC procedure only such as AKTA or other systems e A syringe or manual pump NOTE For best results process all buffers through a 0 45 um filter and degas before use in LC applications B Buffers Native Conditions For your convenience we provide a separate kit containing a set of His60 Ni Extraction Equilibration and Elution Buffers the His60 Ni Buffer Set Cat No 635665 which is sufficient for approximately 20 purifications on 1 ml His60 Ni Superflow Cartridges 5 x 1 ml Cat No 635675 or 5 purifications on 5 ml His60 Ni Superflow Cartridges 5 x 5 ml Cat No 635679 The His60 Ni Buffer Set is also available together with the 1 ml cartridges in the His60 Ni Superflow Cartridge Purification Kit 5 x 1 ml Cat No 635674 and together with the 5 ml cartridges in the His60 Ni Superflow Cartridge Purification K
4. 0 column Section III G Cartridge Regeneration Wash cartridge with 20 column volumes of Equilibration Buffer or wash with 10 column volumes of 20 mM MES 0 3 M NaCl pH 5 0 buffer NOTE Regeneration allows the cartridge to be reused to purify the same protein without significant loss of binding capacity up to 5 times depending on the purification conditions and the target protein Extended Storage over 1 week Wash the cartridge with five column volumes of water after each use Store in 20 ethanol at 4 C Attach supplied bottom cap followed by the top plug D PROTOCOL Complete Regeneration of His60 Ni Superflow Cartridges If you plan to purify multiple proteins using the same column you must use the following resin regeneration protocol before you purify a new protein PT5143 1 030212 Strip Ni ions Wash the cartridge with ten column volumes of 0 2 M EDTA pH 7 0 at room temperature Wash excess EDTA from the cartridges with an additional ten column volumes of double distilled H2O ddH O Charge Resin Add two column volumes of 100 mM NiSQO solution Wash resin to remove excess ions with seven column volumes of ddH O followed by 3 column volumes of 300 mM NaCl and 3 column volumes of ddH O Equilibrate Resin Add ten column volumes of Equilibration Wash Buffer Cartridge is ready to use Resin can be regenerated up to 5 times www clontech com Clontech Laboratories Inc A Takara Bio Company Page 14 of
5. Clontech Laboratories Inc His60 Ni Superflow Cartridges User Manual NOTE FOR FIRST TIME USERS For optimal results please follow the written protocol when performing the first purification Each resin e g Ni IDA Ni NTA Ni TED TALON has a different chemistry Optimal conditions for one resin are not optimal for another resin If you need to modify the protocol please refer to Table Il for compatible reagents and possible effects on the resin PT5143 1 Cat No s 635674 635675 635678 635679 635680 030212 Clontech Laboratories Inc A Takara Bio Company 1290 Terra Bella Avenue Mountain View CA 94043 USA U S Technical Support tech clontech com United States Canada Asia Pacific Europe Japan 800 662 2566 1 650 919 7300 33 0 1 3904 6880 81 0 77 543 6116 His60 Ni Superflow Cartridges User Manual Table of Contents E ee cc se chan os aa ce reste ce clin es ae ee resect ct re ate te he ccs aos nse eae nec once oo ee cece 3 A His60 Ni Supertlow 1 ml amp 5 ml Cartridge Specifications cccscccicescscsactesacsdsstsecsssvedencssacensecctadacsdsidarseessedeaeosascanecctes 3 Be ABON aperon RE pcre eterna csis ste hatte vaciat E E E E E E 4 M TSC OT Ot OS M er E N T ARE 6 Il PRC GO Matenals Fee iC oats cciscipecc sen aA E EE E EEEE E R E A 7 A Eoo e E E N E A E E A E E E er mer errr 7 B Paes Naive CONG OING srs sesccasececocesgacsessataasataiacesetecnsosocossucsocaqeessaseaccospsosocosecoseaaetananmaatc
6. DTA free protease inhibitor Mix gently pipetting up and down several times NOTES e DNase I reduces the viscosity of the lysate allowing for more efficient removal of cellular debris DNase I can be used without lysozyme However if you are treating cells with lysozyme you must also treat these cells with DNase I e Lysozyme helps to fully disrupt bacterial walls and is highly beneficial when extracting high molecular weight proteins gt 40 kDa e The lysozyme solution may form a precipitate Resuspend the contents of the bottle and apply 200 ul of suspension directly to the mix or optionally centrifuge 200 ul of lysozyme solution for 5 min at 14 000 rpm amp use the supernatant to perform the lysis e We recommend that you use our ProteoGuard EDTA Free Protease Inhibitor Cocktail Cat Nos 635672 amp 635673 Incubation Incubate with gentle shaking for 10 min at room temperature If desired you may incubate the solution at 4 C NOTE At the end of the incubation period there should be no visible particles If cell pellet fragments are present resuspend them by pipetting the solution up and down and incubating for an additional 1 2 min www clontech com Clontech Laboratories Inc A Takara Bio Company Page 10 of 15 His60 Ni Superflow Cartridges User Manual 4 Lysate clarification Centrifuge the crude lysate at 10 000 12 000 x g for 20 min at 4 C Carefully transfer the supernatant to a clean tube without d
7. Sample Loading Load the clarified sample onto the cartridge at a flow rate of 0 5 1 ml min and collect fractions PT5143 1 www clontech com 030212 Clontech Laboratories Inc A Takara Bio Company Page 11 of 15 His60 Ni Superflow Cartridges User Manual 5 Wash Step Wash the cartridge using a flow rate of 1 ml min for 1 ml cartridges or 5 ml min for 5 ml cartridges with 8 column volumes of Equilibration Buffer followed by 7 column volumes of Wash Buffer 1 e Equilibration Buffer containing 40 mM imidazole The Wash Buffer is prepared on the LC system by running Pump B at 7 1 Elution Step Elute using a flow rate of 1 ml min for 1 ml cartridges or 5 ml min for 5 ml cartridges with approximately 5 8 column volumes of Elution Buffer containing 300 mM imidazole and collect 1 ml fractions Monitor protein elution by measuring the absorbance of the fractions at 280 nm or performing a Bradford assay Bradford 1976 The collected fractions can be analyzed by SDS PAGE NOTE If necessary for downstream applications remove excess imidazole by gel filtration on a PD 10 column Section III G Cartridge Regeneration Wash cartridge with 20 column volumes of Equilibration Buffer or wash with 10 column volumes of 20 mM MES 0 3 M NaCl pH 5 0 buffer NOTE Regeneration allows the cartridge to be reused to purify the same protein without significant loss of binding capacity up to 5 times depending on the purification conditions an
8. ancaoetosocosoreddeseatananceatansce 7 C Buffers Denaturing Conditions Guanidine HCl or Urea ccccccccsssssseeeeeeeccaaeeseeeecceeeeeaeesseeceeeessaaaaeeeeeeeeeeas 8 D EU YE RCT en meee Oe E eye Tre Nr A Meare enn oer een E eee 8 E Accessories for Automated Purification s ccosesscacssecaccsatccsvacecdsaieaasassiedecesa etescnavaseagiaeaeasansnecsacoaseseecauteaqdeiecesaaaneesesecinnete 8 F Accessories for Syringe Based PUMMCAU OM csicsciscecscecssachteenvasencssrscisdncedavonasael r aaaea Ea r aE eaea raia Kean Aar iinan 8 C UO AM a ser asters os cece ceaa cl soso ceey sedesoce E E E E E E E 8 IV Related Products Extraction Buffers Protease Inhibitors and His Tag Antibodies ccccccsseeeeeeceeeeeeeeeeeeeeees 9 Ve Sampie Pre pail nie Par AU OM serorei nnee nni E E E R 10 A PROTOCOL Extracting Proteins Trom COUS osc crcccecerecsaisuaiseesosssancssvatseutdeeassasecebosasasesaneneuenubendoaaciateiseseaacaaanertenies 10 B PROTOCOL Automated Purification on a Liquid Chromatography System cccccccsssssseeeeeeeeeeeeeseseceeeeeeeeeees 11 C PROTOCOL Manual Purification Using a Syringe eessssseeeesssssssseersssssssseerrssssssseeressssssseerrressssseeereessssseeeeressssse 12 D PROTOCOL Complete Regeneration of His60 Ni Superflow Cartridges cccccccccccccccsseesssssseseeseeeeeeeeeeeeeeeeaaas 14 VE OTS a sa sae E E A E E EEE ees E te ciie sens neaeeoeaunoceced 15 Table of Tables Table 1 His60 Ni S
9. aution the metal ions in His60 Ni Superflow resin Although enough of these ions may remain unaffected to allow protein purification please use it with caution Do at least 20 column volumes of washes preferably with low concentrations of imidazole 40 mM to wash out any reduced metal ions C Not recommended Others MgCl CaCl Ethanol May precipitate proteins causing low yields amp column clogging Glycerol Detergents cannot be easily removed by buffer exchange PT5143 1 www clontech com 030212 Clontech Laboratories Inc A Takara Bio Company Page 5 of 15 His60 Ni Superflow Cartridges User Manual PT5143 1 030212 List of Components Store all components at 4 C His60 Ni Superflow Cartridges 5 x 1 ml Cat No 635675 e 5 His60 Ni Superflow Cartridges 1 ml each e 5 Top Caps e 5 Snap Off End Caps His60 Ni Buffer Set Cat No 635665 e 2x250ml _ His60 Ni Equilibration Buffer e 1x200ml _ His60 Ni Elution Buffer e 1x100ml _ His60 Ni xTractor Buffer NOTE His60 Ni xTractor Buffer is equivalent to the xTractor Buffer supplied in Cat Nos 635623 635625 635656 amp 635671 see Section IV His60 Ni Superflow Cartridge Purification Kit 5 x 1 ml Cat No 635674 e His60 Ni Superflow Cartridges 5 x 1 ml Cat No 635675 e 1 His60 Ni Buffer Set Cat No 635665 His60 Ni Superflow Cartridge 1 x 5 ml Cat No 635680 e His60 Ni Superflow Cartridge 5 ml each e 1 Top Cap e Snap Off End
10. cause overexpressed proteins in prokaryotic systems are sometimes insoluble you may need to purify proteins under denaturing conditions When using high concentrations of guanidine HCl or urea protein unfolding takes place On column refolding or post elution refolding is protein dependent When purifying proteins under denaturing conditions we recommend preparing buffers as indicated below Buffers with 6M Guanidine HCl e Equilibration Buffer 50 mM sodium phosphate 6 M guanidine HCl 300 mM NaCl 20 mM imidazole pH 7 4 e Wash Buffer 50 mM sodium phosphate 6 M guanidine HCl 300 mM NaCl 40 mM imidazole pH 7 4 e Elution Buffer 50 mM sodium phosphate 6 M guanidine HCl 300 mM NaCl 300 mM imidazole pH 7 4 Buffers with 8 M Urea e Equilibration Buffer 50 mM sodium phosphate 8 M urea 300 mM NaCl 20 mM imidazole pH 7 4 e Wash Buffer 50 mM sodium phosphate 8 M urea 300 mM NaCl 40 mM imidazole pH 7 4 e Elution Buffer 50 mM sodium phosphate 8 M urea 300 mM NaCl 300 mM imidazole pH 7 4 NOTE Samples containing guanidine HCl cannot be analyzed by SDS PAGE A buffer exchange to a buffer containing urea must be performed before SDS PAGE analysis Samples containing urea can be analyzed directly by SDS PAGE Enzymes e Benzonase Sigma Cat No E8263 5KU e Recombinant DNase I TaKaRa Cat No 2270A Accessories for Automated Purification e A suitable liquid chromatography system LC procedure only with 1 16 inch t
11. d the target protein Extended Storage over 1 week Wash the cartridge with five column volumes of water after each use Store in 20 ethanol at 4 C Attach supplied bottom cap followed by the top plug C PROTOCOL Manual Purification Using a Syringe PT5143 1 030212 Equilibration Equilibrate the cartridge and all buffers to room temperature or at 4 C Luer Lock Syringe Setup Fill syringe with 5 10 column volumes of Equilibration Buffer www clontech com Clontech Laboratories Inc A Takara Bio Company Page 12 of 15 His60 Ni Superflow Cartridges User Manual PT5143 1 030212 3 Cartridge Setup a Attach the syringe to a Luer Lock Adapter not supplied see Section III F b Remove the top plug from the cartridge add a few drops of buffer from the syringe to the top inlet of the cartridge and attach the syringe to the top of the cartridge via the Luer Lock adapter c Carefully snap off the bottom cap of the cartridge do not twist Equilibrate Cartridge Equilibrate the cartridge with 5 10 column volumes of the Equilibration Buffer at a flow rate of 1 ml min for 1 ml cartridge or 5 ml min for 5 ml cartridge Remove the syringe from the Luer Lock Adapter NOTE For maximum extraction and binding prepare the sample using our xTractor Buffer Section V A If you used incompatible reagents Section I B during the extraction desalt the sample on a PD 10 column Section I G before proceeding to S
12. diminishing the resin s binding capacity Sodium acetate Sodium phosphate Tris Coordinates weakly with metal ions causing a decrease in Up to 50 mM with caution Loss in binding capacity binding capacity can be seen Chelating Agents EDTA EGTA Will strip metal ions from the resin resulting in protein elution Not recommended and a resin color change Denaturing Agents With high concentrations protein unfolding generally takes place Protein refolding on column or after elution is protein dependent With high concentrations protein unfolding generally takes place Protein refolding on column or after elution is protein dependent Detergents CHAPS Ionic detergents such as CHAPS SDS and sarkosy are 1 with caution compatible at concentrations up to 1 Even at low concentrations you should expect interference with binding Ionic detergents such as CHAPS SDS and sarkosy are 1 with caution compatible at concentrations up to 1 Even at low concentrations you should expect interference with binding Triton X 100 Has high absorbance at 280 nm Tween 20 8H Reducing Agents N N Use the resin immediately after equilibrating with buffers 20 mM with caution containing BME Otherwise the resin will change color Do not store the resin in buffers containing BME A slight change in color yellowing of the resin will occur Since DTT is a reducing agent low concentrations will reduce 1 mM with c
13. ding capacity may vary from protein to protein Adaptors may be necessary PT5143 1 www clontech com 030212 Clontech Laboratories Inc A Takara Bio Company Page 3 of 15 His60 Ni Superflow Cartridges User Manual B His60 Ni Superflow Resin PT5143 1 030212 Clontech s His60 Ni Superflow Resin is a high capacity Ni IDA resin that has been optimized for the efficient one step purification of expressed his tagged proteins from bacterial yeast mammalian and baculovirus infected cells The combination of the high density of nickel II ion and the high flow rates of the Superflow 6 agarose beads allow the efficient capture of target his tagged proteins Up to 60 mg of his tagged protein can be adsorbed onto 1 ml of His60 Ni resin data based on purification of ACGFP1 The His60 Ni Superflow Cartridge Purification Kit 5 x 1 ml provides 5 prepacked His60 Ni Superflow Cartridges each containing ml of resin as well as all the buffers needed for protein extraction and purification also available separately as the His60 Ni Buffer Set see Section II The His60 Ni Cartridge Purification Kit 5 x 5 ml provides 5 prepacked His60 Ni Superflow Cartridges each containing 5 ml of resin as well as all the buffers needed for protein extraction and purification also available separately as the His60 Ni Buffer Set see Section IL Limitations of His60 Ni Superflow Resin Please note the following recommendations when using His60 Ni Supe
14. isturbing the pellet This is your starting sample NOTE If the supernatant is not clear centrifuge a second time or filter through a 0 45 um membrane e g cellulose acetate to avoid clogging the IMAC column with insoluble material B PROTOCOL Automated Purification on a Liquid Chromatography System 1 Equilibration Equilibrate the cartridge and all buffers to room temperature or 4 C 2 LC System Set up a Prepare the LC system by filling the tubing with buffer On a binary pump LC system fill Pump A with Equilibration Buffer and Pump B with Elution Buffer b Remove the top plug from the cartridge and start pumping Equilibration Buffer at a flow rate of 1 ml min until a few drops fill in the top inlet c Pause the pump connect the cartridge to the pump outlet and carefully snap off the bottom cap of the cartridge do not twist d Start the pump To avoid introducing air into the system allow a few drops to emerge from the cartridge before connecting to the LC UV monitor inlet port 3 Equilibrate Cartridge Equilibrate the cartridge with 5 10 column volumes of the Equilibration Buffer at a flow rate of 1 ml min for 1 ml cartridge or 5 ml min for 5 ml cartridge NOTE For maximum extraction and binding prepare the sample using our xTractor Buffer Section V A If you used incompatible reagents Section I B during the extraction desalt the sample on a PD 10 column Section I G before proceeding to Step 4 4
15. it 5 x 5 ml Cat No 635678 see Section II The following information is provided if you wish to prepare your own buffers for use with other applications Please note that for FPLC and other automated applications you need to filter the buffers through a 0 45 um filter and degas them before use e Equilibration Buffer 50 mM sodium phosphate 300 mM sodium chloride 20 mM imidazole pH 7 4 e Wash Buffer 50 mM sodium phosphate 300 mM sodium chloride 40 mM imidazole pH 7 4 Wash Buffer is easily made on a binary pump LC system by mixing 7 1 parts of His60 Ni Elution Buffer and 92 9 parts of His60 Ni Equilibration Buffer This buffer ratio can be achieved by running the LC system at 7 1 Pump B Alternatively prepare manually by mixing 710 ul of His60 Ni Elution Buffer with 9 29 ml of His60 Ni Equilibration Buffer e Elution Buffer 50 mM sodium phosphate 300 mM sodium chloride 300 mM imidazole pH 7 4 e Regeneration Buffer 20 mM MES 2 N morpholine ethanesulfonic acid 0 3 M sodium chloride pH 5 0 e Imidazole Use a highly pure low absorbance imidazole ideal for LC applications Fisher Product No BP 305 50 PT5143 1 www clontech com 030212 Clontech Laboratories Inc A Takara Bio Company Page 7 of 15 His60 Ni Superflow Cartridges User Manual C Buffers Denaturing Conditions Guanidine HCl or Urea PT5143 1 030212 Denaturants such as 5 M guanidine HCl or 8 M urea enhance protein solubility Be
16. rflow Cartridges e Do not use chelator containing protease inhibitors or other additives EDTA or strong reducing agents see Table 2 and the note below regarding the use of reducing agents e For automated liquid chromatography LC applications use highly pure low absorbance imidazole Fisher Product No BP 305 50 Always filter buffers through a 0 45 um filter and degas before use e His60 Ni allows protein purification under either native or denaturing conditions The resin is compatible with multiple denaturants and detergents Table 2 NOTE Using BME as a reducing agent with His60 Ni Superflow Resin sharply reduces protein yield If high levels of BME are required for purification we strongly recommend using TALON resin which provides high yields of the target protein in up to 30 mM BME www clontech com Clontech Laboratories Inc A Takara Bio Company Page 4 of 15 His60 Ni Superflow Cartridges User Manual Table 2 Reagent Compatibility with His60 Ni Superflow Resin Based on Literature References Acceptable Concentrations Amino Acids Arginine Glycine Not recommended Glutamine Histidine Binds to His60 Ni and competes with histidine residues in the Can be used at low concentrations 20 his tag mM to inhibit nonspecific binding and at a higher concentration up to 100 mM to elute his tagged proteins Buffers HEPES MOPS Amine groups that are present in these buffers can interact with Ni ions
17. rties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc Your use of this product is subject to compliance with any applicable licensing requirements described on the product s web page at www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Clontech the Clontech logo and TALON are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company 2012 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department PT5143 1 www clontech com 030212 Clontech Laboratories Inc A Takara Bio Company Page 15 of 15
18. tep 5 Sample Loading Fill the syringe with the clarified sample and reconnect it to the Luer Lock Adapter Slowly push the syringe plunger to pass the sample through the cartridge For maximum binding and better yields load the sample at an approximate flow rate of 0 5 1 ml min and collect fractions Wash Step Using a clean syringe wash the resin with 10 column volumes of Wash Buffer at a flow rate of I ml min for 1 ml cartridges or 5 ml min for 5 ml cartridges NOTE If you are using the buffers supplied in the His60 Ni Buffer Set Cat No 635665 the His60 Ni Superflow Cartridge Purification Kit 5 x 1 ml Cat No 635674 or the His60 Ni Superflow Cartridge Purification Kit 5 x 5 ml Cat No 635678 prepare the Wash Buffer by mixing 7 1 parts of Elution Buffer with 92 9 parts of Equilibration Buffer Elution Step Using a clean syringe elute the sample at a flow rate of 1 ml min for 1 ml cartridges or 5 ml min for 5 ml cartridges with approximately five column volumes of Elution Buffer collecting 1 ml fractions Monitor protein elution by measuring the absorbance of the fractions at 280 nm or performing a Bradford assay Bradford 1976 The collected fractions can be analyzed by SDS PAGE www clontech com Clontech Laboratories Inc A Takara Bio Company Page 13 of 15 His60 Ni Superflow Cartridges User Manual NOTE If necessary for downstream applications remove excess imidazole by gel filtration on a PD 1
19. ubing or a binary pump system a quicker and more convenient alternative e Additional connectors and fittings required to attach the cartridges to a Bio Rad BioLogic or a classic FPLC System Section III F Accessories for Syringe Based Purification e Luer Lock Syringe Fittings GE Healthcare Cat No 18 1112 51 for syringe based purification only e M6 FPLC Fittings GE Healthcare Cat Nos 18 1112 58 amp 18 1112 57 for syringe based or automated purification Optional e PD 10 desalting columns GE Healthcare Cat No 17 0851 01 to remove excess imidazole from the final sample when required for downstream applications www clontech com Clontech Laboratories Inc A Takara Bio Company Page 8 of 15 His60 Ni Superflow Cartridges User Manual IV Related Products Extraction Buffers Protease Inhibitors and His Tag Antibodies PT5143 1 030212 xTractor Buffer Kit Cat No 635623 Applications extraction of insoluble protein from inclusion bodies efficient extraction of high molecular weight proteins and complete disruption of bacterial cell walls and membranes e 200 ml xTractor Buffer e 400 ul DNase I e 2 5 ml Lysozyme x Tractor Buffer Cat Nos 635656 635671 amp 635625 Applications bacterial lysis extraction of proteins from yeast cells without the use of glass beads mammalian cell pellet extraction and purification of affinity tagged proteins e 100 ml Cat No 635656 e 250 ml Cat No
20. uperflow Cartridge Specifications ccccccccccccssssssssessseseeeeccccceeeeeeeeaaaaesssseesseeeeeceeeeeeessssaaauaaesssseseeeeeeeess 3 Table 2 Reagent Compatibility with His60 Ni Superflow Resin Based on Literature References cccceeeeeeeeeees 5 Table 3 Troubleshooting Guide for His60 Ni Superflow Cartridges ccccccccccsssssseccecceeaeeesseeececeeeeaeeeeeeceeeeseeaaeeeeeeeeeeas 15 PTS143 1 www clontech com 030212 Clontech Laboratories Inc A Takara Bio Company Page 2 of 15 His60 Ni Superflow Cartridges User Manual I Introduction Clontech s His60 Ni Superflow Cartridges are prefilled with 1 ml or 5 ml of His60 Ni Superflow resin and are ready to use for purification of his tagged proteins using a syringe peristaltic pump or liquid chromatography systems such as AKTA or other FPLC systems A His60 Ni Superflow 1 ml amp 5 ml Cartridge Specifications Table 1 His60 Ni Superflow Cartridge Specifications amt e area Superflow 6 cross linked agarose 2 Binding capacity 60 mg of AcGFPuv 300 mg of AcGFPuv Automated chromatography systems e g AKTA FPLC etc peristaltic pump or syringe Cartridge body material Polypropylene System compatibility The maximum pressure that can be used with the Superflow matrix itself is 10 bar However stability of the cartridges is only guaranteed up to 5 bar High flow rates may lead to reduced recovery of 6xHis tagged protein Bin
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