Protino® 96 Ni-NTA - MACHEREY
Contents
1. Matrix Protino Ni NTA Agarose cross linked 6 beaded agarose Form 50 aqueous suspension containing 30 vol ethanol precharged with Ni Ligand Nitrilotriacetic acid NTA Bead size 45 165 um Bed volume Variable Recommended bed volume 50 uL MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins Table 1 Specifications Protino 96 Ni NTA Binding capacity Up to 2 mg per well when using 50 pL settled agarose Recommended vacuum 0 6 bar depends on sample properties Recommended 3 000 x g depends on sample properties centrifugation speed 2 3 General information Binding capacity The binding capacity of Protino Ni NTA Agarose strongly depends on the characteristics of the polyhistidine tagged protein including amino acid composition molecular weight 3 D structure oligomerization properties etc Protein yield also depends on the total amount and concentration of the target protein in the sample which in turn directly correlate with the expression level and the cell density of the expression culture Furthermore chromatographic conditions may effect protein binding such as sample to resin ratio sample incubation time wash and elution conditions buffer additives etc For optimal results we recommend to use 50 uL of settled Protino Ni NTA Agarose per well A capacity of up to 2 mg per well was determined for the monomeric green fluorescent protein 6 x Hi2. Equilibrate Protino Ni NTA Agarose by adding 500 uL of Lysis Equilibration Buffer NPI 10 to each well Apply vacuum of approximately 0 6 bar for 1 min Allow the buffer to pass the wells Apply vacuum of approximately 0 8 bar for a few seconds to remove any residual fluid from the long drip directors Release the vacuum Batch binding Add up to 750 uL of clarified E coli lysate to the pre equilibrated wells Put a MN Wash Plate upside down into the plate shaker here the MN Wash Plate is used as an adaptor to place the Protino Purification Plate on a plate shaker Place the Protino Purification Plate on top of the inverted MN Wash Plate the drips of the Protino Purification Plate fit right into the holes of the MN Wash Plate Note For robotic applications or more convenient handling MN provides a special frame to fix the Protino Purification Plate on plate shakers see ordering information Depending on the dimension of the shaker fix the plates properly Shake the plate at 20 C for 15 min to 30 min Note Adjust the shaking speed so that the agarose remains in suspension and that no liquid comes out of the wells e g 1 100 rpm for an eppendorf Thermomixer Lysate removal Place the Protino Purification Plate on top of the manifold base Apply vacuum of approximately 0 6 bar until all wells have drained If necessary press down the plate slightly until flow through st
3. Purification of His tag proteins User manual Protino 96 Ni NTA July 2012 Rev 01 MACHEREY NAGEL MN Purification of His tag proteins Table of contents 1 Components 1 1 Contents and storage 1 2 Additional material to be supplied by user 4 2 Product description 5 2 1 The basic principle 5 2 2 Specifications 6 2 3 General information 7 2 4 Compatibility of reagents 9 3 Safety instructions 11 3 1 Risk and safety phrases 11 3 2 GHS classification 11 4 Purification of polyhistidine tagged proteins under native conditions 13 4 1 Preparation of buffers for purification under native conditions 13 4 2 Preparation of cleared E coli lysates under native conditions 14 4 3 Protocol for purification under native conditions using vacuum manifold 15 4 4 Procedure for purification under native conditions by centrifugation 19 5 Purification of polyhistidine tagged proteins under denaturing conditions 20 5 1 Preparation of buffers for purification under denaturing conditions 20 5 2 Preparation of E colilysated under denaturing conditions 22 5 3 Procedure for protein purification under denaturing conditions 22 6 Appendix 23 6 1 Troubleshooting 23 6 2 Ordering information 25 6 3 Product use restriction warranty 26 MACHEREY NAGEL 07 2012 Rev 01 3 Purification of His tag proteins 1 Components 1 1 Contents and storage Protino 96 Ni NTA REF 745425 1 745425 4 Protino Ni NTA Agarose sufficient for 96 preps
4. e g chelating agents EDTA or reducing agents DTT mercaptoethanol see compatibility of reagents section 2 4 Cross contamination The MN Wash Plate prevents the bottom of the Protino Purification Plate to be contaminated by sample or by buffers Any spray will be drained away from the purification plate to waste The risk for cross contamination is highly reduced The MN Wash Plate is discarded after the washing steps 2 4 Compatibility of reagents Table 4 Reagent compatibility chart Reagent Effect Comments Sodium phosphate Used in buffers in order 50 mM is recommended to buffer the solutions at the pH of any buffer should pH 8 be adjusted to 8 although in some cases a pH between 7 and 8 can be used Tris HEPES MOPS Coordinates with Ni Up to 100 mM may be used ions causing a decrease sodium phosphate buffer is in capacity recommended Sodium Chloride Prevents ionic interactions Up to 2 M can be used at and therefore unspecific least 0 3 M should be used binding Imidazole Binds to immobilized Ni Is used at low concentration ions and competes with to reduce non specific binding the polyhistidine tagged 20 mM and to elute the proteins target protein gt 100 mM MACHEREY NAGEL 07 2012 Rev 01 9 Purification of His tag proteins Table 4 Reagent compatibility chart Urea Solubilizes protein Use 8 M for purification under denaturing conditions GuHCl Solubilizes protein Up t
5. Agarose or increase the amount of sample Expression is too low Increase expression level Sometimes the position of the tag influences expression rate and solubility Use a C terminal Histag instead of a N terminal tag or vice versa Increase amount of starting cell material 24 MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins 6 2 Ordering information Product REF Pack of Protino 96 Ni NTA 745425 1 1x96 preps 745425 4 4x 96 preps Protino Ni NTA Agarose 745400 25 25 mL 745400 100 100 mL 745400 500 500 mL Protino Ni NTA Columns 1 mL 745410 5 5 columns Protino Ni NTA Columns 5 mL 745415 1 1 column 745415 5 5 columns Square well Block 740481 4 740481 24 24 Protino Purification Plate 745426 1 1 745426 4 4 MN Shaker Frame 740489 1 Square well Block 740481 4 740481 24 24 Self adhering PE Foil 740676 NucleoVac Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Visit www mn net com for more detailed product information MACHEREY NAGEL 07 2012 Rev 01 25 Purification of His tag proteins 6 3 Product use restriction warranty Protino products components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFI
6. ngerer oder wiederholter Exposition Expositionsweg angeben wenn schl ssig belegt ist dass diese Gefahr bei keinem anderen Expositionsweg besteht Precaution phrases P 210 P 233 P 261 P 272 P 280 P 3024352 P 3334313 P 363 P 4034235 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme heiBen Oberfl chen fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Avoid breathing dust Einatmen von Staub vermeiden Contaminated work clothing should not be allowed out of the workplace Kontaminierte Arbeitskleidung nicht auBerhalb des Arbeitsplatzes tragen Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF ON SKIN Wash with plenty of soap and water Bei Kontakt mit der Haut Mit viel Wasser und Seife waschen If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 12 MACHEREY NAGEL 07 2012 Rev 01 Protino 96 Ni NTA preparation under nativ
7. preparation under native conditions 4 3 Protocol for purification under native conditions using vacuum manifold Setting up the NucleoVac 96 Vacuum Manifold Equilibration Washing steps Elution step Step 4 Place the Protino Purification Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE in the manifold base Final setup Step 4 Place the Protino Purification Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the Square well Block in the manifold Step 1 Insert spacers SQUARE WELL BLOCK in the manifold base Final setup MACHEREY NAGEL 07 2012 Rev 01 15 Protino 96 Ni NTA preparation under native conditions 1 Set up the NucleoVac 96 Vacuum Manifold for the preparation of the Protino Purification Plate Insert spacers labeled MTP MULTI 96 PLATE notched side up into the grooves located on the short sides of the manifold Insert waste container into manifold base Insert the MN Wash Plate on the spacers labeled MTP MULTI 96 PLATE inside the manifold base here the MN Wash Plate is used to avoid cross contaminations Close the manifold base with the manifold lid 2 Prepare Protino Purification Plate Resuspend Pro
8. ED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect
9. HATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Ser
10. and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or 26 MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE W
11. arts Note The vacuum may have to be adjusted for optimal results Apply vacuum of approximately 0 8 bar for a few seconds to remove any residual fluid from the long drip directors When the E coli lysate has passed the plate release the vacuum MACHEREY NAGEL 07 2012 Rev 01 17 Protino 96 Ni NTA preparation under native conditions 6 Washing Wash Protino Ni NTA Agarose by adding 500 uL of Wash Buffer NPI 20 to each well Apply vacuum of approximately 0 6 bar for 1 min Allow the buffer to pass the wells Apply vacuum of approximately 0 8 bar for a few seconds to remove any residual fluid from the long drip directors Release the vacuum Repeat the washing step twice total wash 3 x 500 uL of NPI 20 Remove Protino Purification Plate from the vacuum manifold 7 Set up the NucleoVac 96 Vacuum Manifold for Elution Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Insert spacers labeled SQUARE WELL BLOCK notched side up into the grooves located on the short sides of the manifold Insert a Square well Block into manifold base Close the manifold base with the manifold lid 8 Elution Add 200 pL of Elution Buffer NPI 250 to each well of the Protino Purification Plate Place the Protino Purification Plate on a shaker by using the inverted MN Wash Plate from step 4 batch binding Shake the plate at 20 C and 1 100 rpm for 1 min ep
12. ates 1 mL culture volume 24 well plates 5 mL culture volume or any other appropriate cultivation vessel if larger culture volumes are required Harvest cells by centrifugation at 2 000 x g for 15 min at 4 C Store cell pellets at 20 C or 70 C for at least 1 h 2 Prepare cell extracts Use standard procedures for the preparation of cell extracts such as lysozyme treatment sonication or detergent treatment Note that optimal sample preparation steps have to be determined empirically depending on the characteristics of the of the polyhistidine tagged protein and host organism For preparation of cell extracts from up to 5 mL E coli expression culture we recommend the following protocol as a starting point for further optimization Thaw cell pellets at room temperature Resuspend each pellet in 1 mL of 1 x NPI 10 Buffer containing 0 2 mg mL lysozyme Incubate at room temperature for 30 min in a shaker If the lysate is still viscous add 15 U of Benzonase per well mix and incubate at room temperature for 30 min Benzonase reduces lysate viscosity by rapidly hydrolysing DNA and RNA Centrifuge the crude lysate at 5 000 x g for 30 min at 4 C to remove cellular debris If the supernatant is not clear centrifuge a second time to avoid clogging of the Protino Purification Plate with insoluble material Store supernatant on ice 14 MACHEREY NAGEL 07 2012 Rev 01 Protino 96 Ni NTA
13. ditions using Protino Ni NTA Agarose Individual amounts of the IMAC matrix can be loaded into the wells of the Protino Purification Plate However we recommend to use 50 uL bed volume per well Due to the special designed filter frit material the Protino Purification Plate is absolutely leak free in operation Therefore it is possible to perform the entire purification process equilibration batch binding washing elution directly in the wells of the purification plate Buffer passes through filter frits only by vacuum or centrifugation The Protino Purification Plate enables high well to well reproducibility In addition the long drip directors of the plate minimize cross contamination between the wells The uniquely designed Protino Purification Plate is also ideal for parallel screening experiments to optimize chromatographic conditions including the sample to resin ratio sample incubation time wash and elution conditions buffer additives etc Protino Ni NTA technology Binding of protein is based on the interaction between the polyhistidine tag of the recombinant protein and immobilized Ni ions Protino Ni NTA Agarose consists of the chelating ligand nitrilotriacetic acid NTA immobilized on 6 cross linked agarose beads that are suitable for batch binding gravity flow and FRLC columns The resin is precharged with Ni ions and therefore ready to use Protino Ni NTA Agarose uses NTA which represents the most co
14. e conditions 4 Purification of polyhistidine tagged proteins under native conditions 4 1 Preparation of buffers for purification under native conditions Buffer volumes needed for 96 preps using 50 uL of Protino Ni NTA Agarose per well Step Buffer Volume well Exact Recommended volume plate volume plate Equilibration NPI 10 1x 0 5mL 48 mL 60 mL Washing NPI 20 3x 0 5mL 144 mL 150 mL Elution NPI 250 2x 0 2mL 38 4 mL 50 mL Please note that additional NPI 10 buffer may be needed for the preparation of protein extracts NPI 10 lysis amp equilibration buffer 1 liter 50 mM NaH PO 7 80 g NaH PO 2 H O M 156 01 g mol 300 mM NaCl 17 54g NaCl M 58 44 g mol 10 mM imidazole 0 68 g imidazole M 68 08 g mol Adjust pH to 8 0 using NaOH NPI 20 wash buffer 1 liter 50 mM NaH PO 7 80 g NaH PO 2 HO M 156 01 g mol 300 mM NaCl 17 54 g NaCl M 58 44 g mol 20 mM imidazole 1 36 g imidazole M 68 08 g mol Adjust pH to 8 0 using NaOH NPI 250 elution buffer 1 liter 50 mM NaH PO 7 80 g NaH PO 2 H O M 156 01 g mol 300 mM NaCl 17 54 g NaCl M 58 44 g mol 250 mM imidazole 17 00 g imidazole M 68 08 g mol Adjust pH to 8 0 using NaOH MACHEREY NAGEL 07 2012 Rev 01 13 Protino 96 Ni NTA preparation under native conditions 4 2 Preparation of cleared E coli lysates under native conditions 1 Cultivate and harvest cells E coli cells may be cultivated in 96 well pl
15. ety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol Protino Ni NTA Agarose Ethanol 5 20 x Xn 10 43 37 nickel sulfate lt 0 3 Ethanol 5 20 Nickelsulfat 0 3 Risk phrases R 10 Flammable Entz ndlich R 438 May cause sensitisation by skin contact Sensibilisierung durch Hautkontakt m glich Safety phrases 37 Wear suitable gloves Geeignete Schutzhandschuhe tragen 3 2 GHS classification Only harmful features need not be labeled with H and P phrases until 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard GHS symbol Hazard Precaution contents phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze Protino Ni NTA Agarose Ethanol 5 20 Warning 226 317 210 233 261 nickel sulfate 373 272 280 lt 0 3 3024352 Ethanol 5 20 Achtung 3334313 363 Nickelsulfat 0 3 e 4034235 MACHEREY NAGEL 07 2012 Rev 01 11 Purification of His tag proteins Hazard phrases H 226 H 317 H 373 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen May cause damage to organs through prolonged or repeated exposure Kann die Organe sch digen alle betroffenen Organe nennen sofern bekannt bei l
16. ion of polyhistidine tagged proteins under denaturing conditions Purification of polyhistidine tagged proteins under denaturing conditions is similar to purification under native conditions except that the sample and the buffers contain a denaturant 8 M urea or 6 M guanidine hydrochloride For the preparation of cell extracts and the equilibration steps we recommend to use 6 M guanidine hydrochloride as it is the stronger denaturant For the washing and elution step we recommend to switch to 8 M urea because then eluates can be applied directly to SDS PAGE analysis 5 1 Preparation of buffers for purification under denaturing conditions Buffer volumes needed for 96 preps using 50 uL of Protino Ni NTA Agarose Step Buffer Volume well Exact Recommended volume plate volume plate Equilibration Denaturing 1x 0 5 mL 48 mL 60 mL Lysis Equilibration Buffer Washing Denaturing Wash 3x 0 6 mL 144 mL 150 mL Buffer Elution Denaturing Elution 2x 0 2 mL 38 4 mL 50 mL Buffer Please note that additional Denaturing Lysis Equilibration Buffer may be needed for the preparation of protein extracts Denaturing Lysis Equilibration Buffer 1 liter 0 1 M NaH PO 15 6 g NaH PO 2 HO M 156 01 g mol 0 01 M Tris Base 1 2g Tris Base M 121 14 g mol 6 M guanidine 573 2 g guanidine M 95 53 g mol hydrochloride hydrochloride Adjust pH to 8 0 using NaOH 20 MACHEREY NAGEL 07 2012 Rev 01 Protino 96 Ni NTA preparatio
17. mg shaking for 5 min already leads to acceptable yields However if larger protein amounts are loaded and maximum yield is required we recommend to shake the plate for 15 to 30 min Solubility of the recombinant protein Protein yield is also dependent on solubility of the recombinant protein If proteins are expressed in E coli ideally the target proteins remain soluble in the cytoplasm However proteins that are highly expressed tend to accumulate in insoluble aggregates forming inclusion bodies For solubilization of inclusion bodies buffers containing large amounts of denaturants are used This manual includes instructions for isolation of soluble proteins purification under native conditions see section 4 as well as insoluble proteins from inclusion bodies purification under denaturing conditions see section 5 MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins Improving purity Sometimes optimization of purification procedures is necessary to increase purity Usually lysis equilibration buffers contain 10 mM and the wash buffer 20 mM of imidazole to suppress binding of contaminating proteins To improve specificity increase imidazole concentration In addition for more stringent binding and washing conditions the pH may be reduced from pH 8 closer to pH 7 e g pH 7 4 in all buffers Additives Avoid high concentration of additives that interact with nickel ions and thus reduce capacity
18. mmonly used chelating ligand in IMAC NTA is a tetradentate chelator which occupies four out of the six binding sites in the coordination sphere of the Ni ion The remaining two coordination sites are usually occupied by water molecules and can be exchanged with histidine residues of the recombinant protein Figure 1 This formation of coordination sites has turned out to be optimal for purification of polyhistidine tagged proteins The result is two available binding Sites in the coordination sphere of the Ni ion that enable tight but reversible selective protein interactions Chelation of Ni ions by NTA through four coordination positions minimizes metal leaching during purification and increases specificity for polyhistidine tagged proteins MACHEREY NAGEL 07 2012 Rev 01 5 Purification of His tag proteins r H 0 agarose bead Figure 1 Protino Ni NTA Agarose structure of NTA in complex with Ni 2 2 Specifications Table 1 Specifications Protino 96 Ni NTA Purification plate material Polypropylene Filter frits According to special designed filter frit material the filter plates are absolutely leak free Buffer passes through filter frits only when the water breakthrough pressure is reached by vacuum or centrifugation Filter diameter 8 3 mm Number of wells 96 Volume capacity of well 1 4mL Recommended sample volume Up to 750 pL at 1 100 rpm on an eppendorf Thermomixer
19. n under denaturing conditions Denaturing Wash Buffer 1 liter 0 1 M NaH PO 15 6 g NaH PO 2 HO 0 01 M Tris Base 1 2g Tris Base 8M Urea 480 5 g Urea Adjust pH to 6 3 using HCI M 156 01 g mol M 121 14 g mol M 60 06 g mol Denaturing Elution Buffer 1 liter 0 1 M NaH PO 15 6 g NaH PO 2 H O 0 01 M Tris Base 1 2g Tris Base 8M Urea 480 5 g Urea Adjust pH to 4 5 using HCI M 156 01 g mol M 121 14 g mol M 60 06 g mol MACHEREY NAGEL 07 2012 Rev 01 21 Protino 96 Ni NTA preparation under denaturing conditions 5 2 Preparation of E coli lysated under denaturing conditions 1 Cultivate and harvest cells E colicells may be cultivated in 96 well plates 1 mL culture volume 24 well plates 5 mL culture volume or any other appropriate cultivation vessel if larger culture volumes are required Harvest cells by centrifugation at 2 000 x g for 15 min at 4 C Store cell pellets at 20 C or 70 C for at least 1 h 2 Cellextract preparation For preparation of cell extracts from up to 5 mL E coli expression culture we recommend the following protocol as a starting point for further optimization Thaw cell pellets at room temperature Resuspend each pellet in 0 5 1 mL of 1 x Denaturing Lysis Equilibration Buffer Incubate at room temperature for 30 min in a shaker Centrifuge the crude lysate at 5 000 x g for 30 min at 4 C to remove cellular debris If the s
20. o 6 M can be used B mercaptoethanol Prevents formation of disulfide bonds can reduce Ni ions at higher concentrations Up to 20 mM in samples has been used successfully in some cases DTT DTE Can reduce Ni ions at higher concentrations Up to 10 mM in samples has been used successfully in Some cases Glutathione reduced Can reduce Ni ions at higher concentrations Up to 30 mM in samples has been used successfully in Some cases causing a decrease in capacity Glycerol Prevents hydrophobic Up to 50 can be used interactions between proteins EDTA Coordinates with Ni Not recommended but up to ions causing a decrease 1 mM in samples has been in capacity at higher used successfully in some concentrations cases Ethanol Prevents hydrophobic Up to 20 can be used interactions between ethanol may precipitate proteins proteins causing low flow rates and column clogging SDS Interacts with Ni ions Not recommended but up to 0 396 in samples has been used successfully in some cases Triton Tween etc Nonionic detergents Removes background proteins Up to 296 can be used 10 MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins 3 Safety instructions The following components of the Protino 96 Ni NTA products contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 3 4 Risk and saf
21. pendorf Thermomixer Place the Protino Purification Plate on top of the manifold base Elute the polyhistidine tagged proteins by applying vacuum of approximately 0 6 bar for 1 min Apply vacuum of approximately 0 8 bar for a few seconds to remove any residual fluid from the long drip directors Release the vacuum Repeat the elution step once or until all target protein is removed You may collect the eluates either in the same Square well Block or in fractions by changing the block between each elution step Store eluted protein on ice Note Prior further analysis mix the eluate thoroughly 18 MACHEREY NAGEL 07 2012 Rev 01 Protino 96 Ni NTA preparation under native conditions 4 4 Procedure for purification under native conditions by centrifugation Follow the standard protocol as described in section 4 2 The vacuum steps are substituted by centrifugation of the Protino Purification Plate at 3 000 x g for 2 min at RT During all centrifugation steps the Protino Purification Plate should be placed on a Square well Block see ordering information to collect the waste Omit the MN Wash Plate During the elution step the Protino Purification Plate is placed either on top of a Rack of MN Tube Strips see ordering information or on a Square well Block MACHEREY NAGEL 07 2012 Rev 01 19 Protino 96 Ni NTA preparation under denaturing conditions 5 Purificat
22. s GFPuv 32 kDa expressed in E coli Incubation time Especially during sample incubation sufficient agitation is necessary to disperse the agarose beads We recommend to use 50 uL of settled Protino Ni NTA Agarose per well an agitation speed of 1 100 rpm eppendorf Thermomixer and a sample volume of up to 750 uL As a starting point we recommend to shake the suspension for 15 30 min see Figure 2 However as the purification plate is absolutely leak free longer sample incubation times can be used to achieve maximum yield see Figure 2 If shorter incubation periods are required e g according to protein stability incubation can be reduced e g to 5 min but then yield may decrease see Figure 2 1 Binding capacity will vary for each polyhistidine tagged protein and strongly depends on chromatographic conditions MACHEREY NAGEL 07 2012 Rev 01 T Purification of His tag proteins Yield mg well 0 0 ot 0 0 0 5 1 0 1 5 2 0 2 5 3 0 Loaded protein per mg well Figure 2 Impact of sample incubation time on protein yield Protino Ni NTA Agarose 50 uL bed volume was loaded with increasing amounts of His tagged Green Fluorescent Protein 6xHis GFPuv in a total volume of 750 uL of NPI 10 After washing with 1 5 mL of NPI 20 the target protein was eluted with 2 x 200 uL of NPI 250 Yields of 6xHis GFPuv are plotted versus the amount of loaded protein When wells are loaded with up to 0 8
23. sure that all additives are compatible see compatibility of reagents 2 4 Protein Incorrect buffer composition elutes with Check composition and pH of all buffers wash buffer Protein does not elute Elution conditions are too mild Increase concentration of imidazole from 250 mM to 500 mM Protein has precipitated Elute under denaturing conditions MACHEREY NAGEL 07 2012 Rev 01 23 Purification of His tag proteins Problem Possible cause and suggestions Protein does not elute continued Unwanted proteins elute with poly histidine tagged protein Insufficient wash Use larger volumes for washing step Use NPI 50 for third washing step containing 50 mM imidazole Binding and wash conditions are too mild Use 10 20 mM imidazole in the binding and washing buffers Contaminating proteins and target protein are linked together via disulfide bonds Add up to 20 mM 2 mercaptoethanol to reduce disulfide bonds Contaminating proteins are proteolytic products of target protein Perform cell lysis at 4 C Include protease inhibitors Resin is not saturated with His tagged protein Contaminating host proteins have a better chance to bind to the resin when only small amounts of target protein are present in the lysate Very low amounts of polyhistidine tagged protein are not able to replace the majority of contaminating proteins effectively Reduce the amount of Protino Ni NTA
24. tino Ni NTA Agarose by mixing thoroughly to achieve a homogeneous suspension Pour the entire contents of one bottle 11 mL of suspension into a appropriate tray Add 44 mL of deionized water to get a final volume of 55 mL Gently agitate the tray to achieve a homogeneous suspension Note Proper agitation is necessary to make sure the agarose beads are evenly mixed If the suspension is not mixed well agarose beads settle to the bottom of the tray and lead to inconsistent filling and finally to poor well to well reproducibility Use a tray with a flat bottom Keep mixing the agarose suspension until all wells of the plate are filled The suspension is considered homogeneous if it appears uniform to the eye No clouds or settled agarose beads should be visible Dispense 500 uL of the suspension into each well of the Protino Purification Plate 500 uL of 1096 suspension corresponds to 50 uL bed volume Note If you do not use all wells of the plate for purification seal the top of the empty wells with a foil Place the Protino Purification Plate on top of the manifold base Apply vacuum of approximately 0 6 bar for 1 min If necessary press down the plate slightly until flow through starts When the diluted storage solution of the agarose has passed the plate release the vacuum 16 MACHEREY NAGEL 07 2012 Rev 01 Protino 96 Ni NTA preparation under native conditions Equilibration
25. upernatant is not clear centrifuge a second time to avoid clogging of the Protino Purification Plate with insoluble material Note that optimal sample preparation steps have to be determined empirically depending on the characteristics of the of the polyhistidine tagged protein and host organism 5 3 Procedure for protein purification under denaturing conditions Proceed to section 4 3 vacuum manifold or 4 4 centrifugation with the following modifications Use Denaturing Lysis Equilibration Buffer for the equilibration and washing step Use Denaturing Elution Buffer for elution 22 MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Sample lysate contains insoluble material Wells of the Use centrifugation or filtration to avoid clogging plate have become f clogged Sample lysate remains viscous from genomic DNA Add additional DNase Protein does Problems with vector construction Ensure that protein and tag are in frame Sometimes the position of the tag influences expression rate and solubility Evaluate N and C terminally tagged variants of the protein His Tag is not accessible not bind to Use denaturing conditions to purify the protein the resin Use a C terminal Histag instead of a N terminal tag or vice versa Incorrect binding conditions e Check composition and pH of all buffers En
26. using an agarose bed 5 5 mL 4x5 5mL volume of 50 pL for the purification of up to 2 mg polyhistidine tagged protein Protino Purification Plate proprietary leak free purification plate for 1 4 retaining the chromatographic resins MN Wash Plate one plate is used to minimize cross contamination the other plate is used as 2 8 an adaptor to place the Protino Purification Plate on a plate shaker User manual 1 1 Shipping and storage The product is shipped at ambient temperature Upon receipt Protino Ni NTA Agarose suspension should be stored at 2 8 C and is stable up to 1 year Do not freeze All other components can be stored at ambient temperature 1 2 Additional material to be supplied by user Plate shaker Vacuum manifold system OR centrifuge Plate s for collecting eluate fraction s Square well Block see ordering information For the purification under native conditions prepare the buffers according section 4 1 For the purification under denaturing conditions prepare buffers according section 5 1 4 MACHEREY NAGEL 07 2012 Rev 01 Purification of His tag proteins 2 Product description 2 1 The basic principle Overview Protino Ni NTA products enable convenient high throughput purification of recombinant polyhistidine tagged proteins by immobilized metal ion affinity chromatography IMAC Proteins from any expression system can be purified under native or denaturing con
27. vice Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks Benzonase is a registered trademark of Merck KGaA eppendorf Thermomoxer is a registered trademark of Eppendorf AG Germany Protino is a registered trademark of MACHEREY NAGEL All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 07 2012 Rev 01 27
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