Ni-IDA Resin
Contents
1. Purification of Polyhistidine Tagged Proteins User manual Protino Ni IDA 150 packed columns Protino Ni IDA 1000 packed columns Protino Ni IDA 2000 packed columns Protino Ni IDA Resin Protino Multi 96 Ni IDA June 2006 Rev 03 MACHEREY NAGEL MN Purification of Polyhistidine Tagged Proteins Table of contents 1 Contents 5 2 Introduction 7 2 1 The basic principle 7 2 2 About this user manual 8 3 Product description 10 3 1 Specifications 10 3 2 Purification under native and denaturing conditions 11 3 3 Binding capacity of Protino Ni IDA 12 3 4 Culture size 15 3 5 Binding washing and elution 19 3 6 Buffer compositions 20 3 7 Materials to be supplied by the user 22 3 8 Protino Columns for gravity flow column chromatography using Protino Ni IDA Resin 23 3 9 Compatibility of reagents 24 3 10 Storage conditions 25 4 Safety instructions risk and safety phrases 26 5 Purification of polyhistidine tagged proteins from E coli under native conditions 27 5 1 Preparation of cleared lysates under native conditions 27 5 2 Protino Ni IDA packed columns purification under native conditions 28 5 3 Protino Ni IDA Resin gravity flow column chromatography under native conditions 31 5 4 Protino Ni IDA Resin batch gravity flow column chromatography under native conditions 32 5 5 Protino Ni IDA Resin batch purification protocol under native conditions 33 5 6 Protino Ni IDA Resin medium pressure c2. IDA is a threedentate chelator which occupies three of the six binding sites in the coordination sphere of the Ni ion The remaining three coordination sites are usually occupied by water molecules and can be exchanged with histidine residues of the recombinant protein Figure 1 kat o Ge oo E ER HO Silica Bead sar H O Figure 1 Protino Ni IDA Structure of IDA in complex with Ni MACHEREY NAGEL 06 2006 Rev 03 7 Purification of Polyhistidine Tagged Proteins 2 2 About this user manual For quick orientation in this user manual please follow the corresponding cross reference given below Product Application Page s Protino Ni IDA packed columns Gravity flow column chromatography 28 38 Protino Ni IDA Resin Gravity flow column chromatography 131 39 Batch binding in combination with 32 gravity flow column chromatography Batch purification 33 Medium pressure column 34 chromatography FPLC Protino Multi 96 Ni IDA Gravity flow column chromatography 37 Protino Ni packed columns Experienced users who are performing the purification of His tagged proteins using Protino Ni packed columns may refer to the Protocol at a glance instead of this user manual see section 5 2 1 The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the
3. Solubilization of inclusion bodies Refer to section 6 1 For detailed information on culture and buffer volumes for cell extract preparation also see Table 2 section 3 4 Column equilibration Equilibrate Protino Ni IDA packed columns with Denaturing Solubilization buffer Allow the column to drain by gravity Protino Ni IDA 150 1000 packed columns are designed to fit into most 15 ml conical centrifuge tubes e g BD Falcon Cat No 352097 for convenient fraction collection Binding Add the supernatant cleared lysate see section 6 1 to the pre equilibrated column and allow the column to drain by gravity Washing Wash the column with Denaturing Solubilization buffer Allow the column to drain by gravity Elution Elute the polyhistidine tagged protein in a new collecting tube by adding Denaturing Elution Buffer Allow the column to drain by gravity Note Depending on protein characteristics 90 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 41 Protino Ni IDA 6 3 Protino Ni IDA Resin gravity flow column chromatography under denaturing conditions For column IMAC using Protino Ni IDA Resin we generally recommend the gravity flow procedure This protocol describes gravity flow column chromatography using Pr
4. 800 ug 5 mg 10 mg 6xHis GFPuv 32kDa 40 mg resin 250 mg resin 500 mg resin Application Gravity flow columns Physical form Ready to use columns filled with dry matrix precharged with Ni Specifications Protino Ni IDA Resin Protein capacity 20 mg g resin 6xHis GFPuv 32kDa 10 mg ml bed volume Application Batch Gravity flow column FPLC Physical form Dry matrix precharged with Ni Max pressure 1450 psi 100 bar Specifications Protino Multi 96 Ni IDA Protein capacity 1 mg well 6xHis GFPuv 32kDa 50 mg resin well Application Gravity flow chromatography Physical form Ready to use 96 well plates filled with dry matrix precharged with Ni Specifications Protino Ni IDA packed columns Resin and Multi 96 kits Matrix Macroporous silica Density 0 5 g ml 1 g resin corresponds to 2 ml bed volume Chelating group IDA iminodiacetic acid The nomenclature of the Protino packed columns is independent from the binding capacity but is to reflect the dimension of the column such as small medium and large 10 MACHEREY NAGEL 06 20009Rev 03 Purification of Polyhistidine Tagged Proteins Specifications Protino Ni IDA packed columns Resin and Multi 96 kits Mean particle size 90 um pH stability 3 7 5 22h 2 30r7 5 8 5 x2h Storage lt 25 C Recommended imidazole 0 mM concentration for load wash Recommended imidazole lt 250 mM concentration for elution e Prot
5. Elution buffer according to the instruction given in this section Note Due to the dissociation of urea prepare buffers immediately prior to use Lysis Equilibration Wash buffer LEW buffer 1 liter e 50mM NaH PO 7 8 g NaH PO x 2 H2O MW 156 01 g mol e 300 mM NaCl 17 5 g NaCl MW 58 44 g mol Adjust pH to 8 0 using NaOH Denaturing Solubilization buffer 1 liter e 50 mM NaH PO 7 8 g NaH 2PO x 2 H2O MW 156 01 g mol e 300 mM NaCl 17 5 g NaCl MW 58 44 g mol e 8Murea 480 5 g MW 60 06 g mol Adjust pH to 8 0 using NaOH Denaturing Elution buffer 1 liter e 50mM NaH PO 7 8 g NaH PO x 2 H2O MW 156 01 g mol e 300 mM NaCl 17 5 g NaCl MW 58 44 g mol e 8Murea 480 5 g MW 60 06 g mol e 250 mM imidazole 17 0 g imidazole MW 68 08 g mol Adjust pH to 8 0 using NaOH MACHEREY NAGEL 06 2006 Rev 03 21 Purification of Polyhistidine Tagged Proteins 3 7 Materials to be supplied by the user Appropriate centrifuge sonicator e Appropriate centrifugation collection tubes Lysozyme e Protino Ni IDA 150 1000 2000 packed columns kits Purification under native conditions Kits already contain buffer stock solutions that have to be prepared according to the instructions section 5 2 1 Purification under denaturing conditions Denaturing Solubilization buffer Denaturing Elution buffer additional LEW buffer sodium phosphate sodium chloride urea and imidazole For bu
6. For maximum yield use an excess amount of polyhistidine tagged protein in the loaded sample E g apply up to 75 mg of anticipated 6xHis GFPuv per 1 g of Protino Ni IDA Resin For maximum recovery use up to 20 mg of 6xHis GFPuv per 1 g of Protino Ni IDA Resin The concentration of the polyhistidine tagged protein in the culture may vary from lt 1 mg l up to 200 mg l depending on cell density and expression level It is recom mended to determine the protein concentration for each expression experiment e g via SDS PAGE On average 250 ml of culture will produce approximately 1 g of pelleted wet cells Transfer the cell lysate from a 200 750 ml high expression at 100 mg l or 2000 7500 ml low expression at 10 mg l E coli culture to 1 g of Protino Ni IDA Resin n order to obtain highly concentrated lysates lyse wet cells in 2 5 ml LEW buffer per 1 g wet mass The volume of LEW buffer should be adjusted according to the amount of polyhistidine tagged protein in the culture For example 1 g cells may be resuspended in 2 5 ml LEW buffer if a protein is expressed at 50 200 mg l For cultures with lower target protein content 1 g cells should be resuspended in 2 ml of LEW buffer For recovering polyhistidine tagged protein from E coli cultures we recommend treatment with lysozyme in combination with sonication If you are purifying recombinant protein from eukaryotic cells treat the cells with an appropriate buffer containing
7. 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 of the IMAC column with insoluble material Store supernatant on ice Proceed to section 5 2 5 3 5 4 5 5 5 6 or 5 7 MACHEREY NAGEL 06 2006 Rev 03 27 Protino Ni IDA 5 2 Protino Ni IDA packed columns purification under native conditions 5 2 1 Protocol at a glance Note This section only refers to Protino Ni IDA 150 1000 and 2000 packed columns Protino Ni IDA packed columns 1 Cultivate and 4 500 6 000 xg 4 500 6 000xg 4 500 6 000 xg harvest cells 15 min at 4 C 15 min at 4 C 15 min at 4 C 2 Preparation of working solutions per column 8x LEW buffer 0 3 ml 1 5 ml 2 5 ml water 2 1 ml 10 5 ml 17 5 ml 1x LEW buffer 2 4ml 12ml 20 ml 4x Elution buffer 0 25 ml 1 5 ml m water 0 75 ml 4 5 ml 9ml 1x Elution buffer 1ml 6 ml 12ml 3 Cell Extract Preparation Refer to section 5 1 4 Column Equilibration 1x LEW buffer 320 yl 2 ml 4 mi 9 pinang Load clarified Load clarified Load clarified e lysate onto the lysate onto the lysate ontothe column column column I 6 Washing 1x LEW buffer zzu EN l 7 Elution 1x Elution buffer 3 x 240 yl 3x 1 5 ml 28 MACHEREY NAGEL 06 2006 Rev 03 5 2 2 Protino Ni IDA packed col
8. MACHEREY NAGEL 06 2006 Rev 03 5 Purification of Polyhistidine Tagged Proteins 1 Contents continued Protino Ni IDA Resin Cat No 745210 5 745210 30 745210 120 745210 600 Protino Ni IDA Resin 5g 30g 120g 600 g Protocol 1 1 1 1 Protino Multi 96 Ni IDA 1 x 96 preps 4 x 96 preps Cat No 745300 1 745300 4 Protino Multi 96 Ni IDA Plate 1 4 8x LEW buffer 70 ml 210 ml 4x Elution buffer 30 ml 115 ml Protocol 1 1 6 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 2 Introduction 2 1 The basic principle Protino Ni IDA products enable fast and convenient purification of recombinant polyhistidine ta agged proteins by immobilized metal ion affinity chromatography IMAC Protino Ni IDA is a dry silica based resin precharged with Ni ions Binding of protein is based on the interaction between the polyhistidine tag or the recombinant protein and immobilized Ni ions The chelating group of Protino Ni IDA is based on IDA iminodiacetic acid which enables strong and efficient binding of target protein onto the IMAC matrix Figure 1 In contrast to traditional IDA matrices Protino Ni IDA shows an optimized low density of IDA ligands which is created by a special manufacturing process This non saturating surface concen tration of IDA eliminates non specific interactions of contaminating proteins with the adsorbent As a result Protino Ni IDA ensures higher target protein purity
9. contain s the majority of the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 31 Protino Ni IDA 5 4 Protino Ni IDA Resin batch gravity flow column chromatography under native conditions Although we recommend gravity flow procedure polyhistidine tagged proteins may be purified by the following batch gravity flow protocol Note Usually the yield is not significantly increased using time consuming shaking 1 Batch binding Add the appropriate amount of Protino Ni IDA Resin directly to the cleared lysate see section 5 1 Gently mix the material on an orbital shaker for 5 15 min Do not use a magnetic stirrer to avoid generating fine particles through excessive physical force The amount of resin required depends on the amount of polyhistidine tagged protein to be purified The binding capacity of Protino Ni IDA Resin varies from protein to protein See section 3 3 for general guidelines 1 g of Protino Ni IDA Resin will result in 2 ml bed volume The length of time required for optimal binding will vary from protein to protein 2 Transfer Transfer the lysate resin mixture to an empty chromatography column e g Protino Columns see section 3 7 and ordering information Let the resin settle by gravity flow 3 Washing Wash the column with 8 bed volumes of LEW buffer Allow the column to drain by gravity 4 Elution Elute the polyhistidine tagged protein in three fractions Add 3 x 3 bed volumes
10. 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 Service 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 Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail tech bio mn net com 48 MACHEREY NAGEL 06 2006 Rev 03
11. mi a 4 4 8 1 6 T D 27 15 15 30 6 c o o 0 16 16 80 16 o tc 60 60 300 60 a T 0 2 0 2 0 4 0 08 TE z SEG 0 6 0 6 1 2 0 24 2 30 SooE 0 64 0 64 3 2 0 64 ono a 2 4 2 4 12 2 4 L 1 1 2 0 4 9o0 z cqQ 15 15 7 5 24 e m E a5 3 LOGE 4 4 20 4 e oo 9e Uu a x 24 24 75 24 do 5 5 4 0 8 29042 Zoer 30 30 15 4 8 o 253 E gotE 8 8 40 8 o6 oo fa 8 a 2 48 48 150 48 1 Lysis Equilibration Wash buffer Denaturing Solubilization buffer Inclusion Bodies MACHEREY NAGEL 06 2006 Rev 03 17 Purification of Polyhistidine Tagged Proteins Figure 4 Required culture volumes and LEW buffer volumes for maximum protein recovery or maximum yield in dependence on protein expression level The volumes noted below are only exemplary and are shown for 1 g of Protino Ni IDA Resin purification under native conditions Please use these recommen dations as a starting point to evaluate optional purification results Note that purification conditions have to be optimized for each individual polyhistidine tagged protein 1 g Protino Ni IDA Resin N for maximum recovery for maximum yield load 20 mg protein load 75 mg protein high expression low expression high expression low expression culture culture culture culture 100 mg l 10 mg l 100 mg l 10 mg l use 200 ml use 2000 ml use 750 ml use 7500 ml culture culture culture culture 70 8 g cell 8
12. on binding capacity please also refer to sections 3 3 and 3 4 Table 3 Protino Columns to be used with Protino Ni IDA Resin Volume capacity Max amount of Protein binding ml Protino Ni IDA capacity Resin per column mg g Protino Columns 14 ml 14 1 4 28 Protino Columns 35 ml 35 3 5 70 1 Protein binding capacity refers to 6xHis GFPuv MACHEREY NAGEL 06 2006 Rev 03 23 Purification of Polyhistidine Tagged Proteins 3 9 Compatibility of reagents Buffer components that chelate metal ions such as EDTA and EGTA should not be used since they strip Ni ions from the matrix Do not use buffers with pH 8 4 since silica dissolves in solutions of high pH Table 4 Reagent compatibility chart Reagent Effect Comments Sodium phosphate Used in LEW and Elution buffer in order to buffer the solutions at pH 8 50 mM is recommended The pH of any buffer should be adjusted to 8 although in some cases a pH between 7 and 8 can be used Tris Coordinates with Ni ions causing a decrease in capacity 10 mM may be used sodium phosphate buffer is recommended Sodium Chloride Prevents ionic interactions and therefore unspecific binding Up to 2 Mcan be used at least 0 3 M should be used Binds to immobilized Ni ions and competes with Should not be included in LEW unigazele the polyhistidine tagged buffer proteins Urea Solubilizes protein mat dedu uncer
13. 6 SDS 5 bed volumes of deionized water 5 bed volumes of 100 EtOH 5 bed volumes of deionized water 5 bed volumes of 100 mM EDTA pH 8 5 bed volumes of deionized water 5 bed volumes of 100 mM NiSO 10 bed volumes of deionized water MACHEREY NAGEL 06 2006 Rev 03 43 Purification of Polyhistidine Tagged Proteins 8 Appendix 8 1 Troubleshooting Problem Possible cause and suggestions Sample lysate contains insoluble material e Ifthe sample is not clear use centrifugation or filtration 0 45 um membrane to avoid clogging of the IMAC column Sample does not enter column bed Sample lysate contains genomic DNA Lysate may remain viscous from incomplete shearing of genomic DNA after sonication Add 5 ug ml DNase and incubate on ice for 10 min Problems with vector construction Ensure that protein and tag are in frame Protein does not Incorrect binding conditions bind to the resin T e Check composition of buffers and verify pH 7 8 Ensure that there is no chelating or strong reducing reagent or imidazole present Incorrect buffer composition Protein elutes with Check composition of buffers and verify pH 7 8 Ensure wash buffer that there are no chelating or strong reducing agents or imidazole present Protein does not Elution conditions are too mild elute from column e Increase concentration of imidazole Insufficient wash Use larger volumes for washing step Unwanted proteins Binding
14. Figure 2 Binding behaviour of 6xHis GFPuv to Protino Ni IDA Resin Gravity flow columns packed with 40 mg of Protino Ni IDA Resin were loaded with increasing volumes of an E colilysate containing 6xHis GFPuv protein concentration 2 mg ml After washing with 640 ul LEW buffer the target protein was eluted with 960 ul Elution buffer Yield left axis and recovery right axis of 6xHis GFPuv are plotted versus the amount of loaded protein For convenient analysis the values are converted to mg 6xHis GFPuv per 1 g resin MACHEREY NAGEL 06 2006 Rev 03 13 Purification of Polyhistidine Tagged Proteins Recovery rates and yield can be increased by using samples containing higher concentrated polyhistidine tagged protein 6xHis GFPuv Figure 3 shows that the yield of purified polyhistidine tagged protein is not only depending on the total amount of target protein loaded on the column also see Figure 2 but also on its concentration in the lysate Consequently the concentration of target protein in the sample should be as high as possible 50 40 30 eluted 6xHis GFPuv mg g 20 A 0 5 mg ml 2 0 mg ml m 8 0 mg ml 0 T 7 ae a a a a a T T oci 0 10 20 30 40 50 60 70 80 loaded 6xHis GFPuv mg g Figure 3 Binding behaviour of 6xHis GFPuv to Protino Ni IDA at different concentrations of the polyhistidine tagged protein in the sample Recombinant 6xHis GFPuv was expressed in E coli The concentration of th
15. GuHCI Solubilizes protein Up to 6 M can be used p mercaptoethanol Prevents formation of disulfide bonds Can reduce Ni ions at higher concentrations Up to 50 mM in samples has been used successfully in some cases Can reduce Ni ions at Up to 10 mM in samples has DTT DTE higher concentrations been used successfully in some cases do Up to 30 mM in samples has Glutathione Can reduce Ni ions at reduced higher concentrations PPE MUSEO SHEEPS SIINSES cases Prevents hydrophobic Glycerol interactions between Up to 50 can be used proteins 24 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins Table 4 Reagent compatibility chart Reagent Effect Comments Coordinates with Ni ions causing a decrease Not recommended but up to 1 EDTA mM in samples has been used neapaciy a higher successfully in some cases concentrations O E Prevents hydrophobic ES ae e can be a Ethanol interactions between Manor May preeipirate proteins causing low flow rates proteins and column clogging Interacts with Ni ions Not recommended but up to SDS causing a decrease in 0 5 96 in samples has been used capacity successfully in some cases Removes background Triton Tween proteins Up to 2 can be used 3 10 Storage conditions All kit components can be stored at room temperature 20 25 C and are stable up to one year MACHEREY NAGEL 06 2006 R
16. a mild detergent Sambrook et al 1989 MACHEREY NAGEL 06 2006 Rev 03 15 Purification of Polyhistidine Tagged Proteins Table 2 Determination of culture and buffer volume requirements concen results in amount of recommen recommen tration of protein load ded E coli ded E coli HisTag culture pellet wet protein in volume mass the culture mg ml g E high recovery max 20 200 0 8 Se 7100mg ieldua 75 750 3 e S 8 low recovery max 20 2000 8 a 1O mg vieldinax 75 7500 30 vc E high reCOVETY max 0 8 8 0 04 On ZSE 100 mg l vieldmax 3 30 0 12 o 23 o 5 2 9 low recovery max 0 8 80 0 32 E Svim vieldmar 3 300 12 a o high recovery max 5 50 0 2 TODy 2 7 100mgl yieldmax 18 8 188 0 75 e O30 oE reCOVETY max 5 500 2 S Go low o Q 10mgl 3 high reCOVErY max 10 100 0 4 ILo s z td 100 mg l vieldmax 37 5 375 1 5 S a5 o E 8 o S iow reCOVETY max 10 1000 4 gs a Oman Dauid 37 5 3750 15 On average 250 ml of culture will produce approximately 1 g of pelleted wet cells 16 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins Table 2 Determination of culture and buffer volume requirements continued native conditions denaturing conditions LEW buffer LEW buffer LEW buffer DS buffer resuspension resuspension washing of IB lysis of IB mi mi mi
17. and wash conditions are too mild elute with polyhistidine tagged Add small amounts of imidazole 1 10 mM Verify that the protein imidazole concentration is low enough to bind the polyhistidine tagged protein 44 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins Problem Possible cause and suggestions Contaminating proteins and target protein are linked together via disulfide bonds e Add up to 30 mM f mercaptoethanol to reduce disulfide bonds Contaminating proteins are proteolytic products of target protein Perform cell lysis at 4 C Unwanted proteins Include protease inhibitors elute with e polyhistidine tagged Expression is too low protein continued 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 Increase expression level Increase amount of starting cell material Do not exceed recommended lysis volumes MACHEREY NAGEL 06 2006 Rev 03 45 Purification of Polyhistidine Tagged Proteins 8 2 Ordering information Product Cat No Pack of Protino Ni IDA Resin 745210 5 5g Protino Ni IDA Resin 745210 30 30g Protino Ni IDA Resin 745210 120 120g Protino Ni IDA Resin 745210 600 600 g Protino Ni IDA 150 packed columns contains 40 mg of res
18. ant the supernatant and dispose of it Repeat the washing step one or two more times total wash 2 3 x 4 bed volumes of LEW buffer 4 Elution Add 3 bed volumes of Elution buffer and mix for 5 min Sediment the resin by gravity or centrifugation at 500 x g for 1 min Carefully decant or pipette the eluate in a new tube Repeat the elution step two more times total elution 3 x 3 bed volumes of LEW buffer Note Depending on protein characteristics 9096 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 33 Protino Ni IDA 5 6 Protino Ni IDA Resin medium pressure column chromatography under native conditions According to the physical stability of the Protino Ni IDA Resin it is ideally suited for medium pressure column chromatography The rigid matrix can be run under high flow rates and high back pressure Furthermore Protino Ni IDA Resin does not shrink or swell upon hydration As a starting point run columns at flow rates of 0 5 1 0 ml min cm If the polyhistidine tagged protein does not bind further reduce the flow rate Optimal flow rates have to be determined empirically because dissociation rates vary widely from protein to protein 1 34 Column preparation slurry packing Make a 10 96 w v slurry of Protino Ni IDA Res
19. e For preparation of cell extracts from large volumes of E coli expression culture gt 5 ml culture refer to section 5 1 MACHEREY NAGEL 06 2006 Rev 03 Protino Ni IDA Cell extracts from lt 5 ml E coli expression culture 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 1x LEW 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 Multi 96 Ni IDA Plate with insoluble material Store supernatant on ice Equilibration Equilibrate the Protino Multi 96 Ni IDA Plate by adding 500 ul of 1x LEW buffer to each well Allow the wells to drain by gravity Binding Add the cleared lysate supernatant see step 3 or section 5 1 to the pre equilibrated wells of the Protino Multi 96 Ni IDA Plate Allow the wells to drain by gravity Note The lysate should not contain any imidazole since most proteins do not bind to the resin in the presence of e
20. e target protein in the culture reached 100 mg l 1 g cells were lysed in 2 ml LEW buffer according to section 3 4 in order to obtain a highly concentrated lysate The concentration of 6xHis GFPuv in the lysate was 8 mg ml Gravity flow columns packed with 40 mg of Protino Ni IDA Resin were loaded with increasing volumes amounts of the lysate After washing the target protein was eluted with Elution buffer The yield of 6xHis GFPuv is plotted against the amount of loaded target protein The same test was performed using diluted lysates with concentrations of 0 5 and 2 mg ml For convenient analysis the values are converted to mg 6xHis GFPuv per 1 g resin Please note The higher the protein concentration in the sample and the higher the total amount of protein loaded on Protino Ni IDA packed columns or Resin the higher will be the absolute yields For example if loading 75 mg 6xHis GFPuv concentration 8 mg ml per g Protino Ni IDA Resin a maximum yield of 50 mg g can be obtained 14 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 3 4 Culture size As outlined above the protein yield depends on various parameters However some recommendations on protein load and culture size can be given as a starting point Note that yield and protein load are exemplified for the 6xHis GFPuv 32 kDa and may vary from protein to protein e Use rather high concentrations of the target protein in the sample
21. ed 740563 1 columns MN Tube Strips 740637 5 sets 8 3 Product use restriction warranty Protino Ni IDA products were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of Protino Ni IDA for a specific application range as the performance characteristic of this kit has not been verified to a specific organism 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 an extra copy MACHEREY NAGEL does not warrant against 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 against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or produc
22. ev 03 25 Purification of Polyhistidine Tagged Proteins 4 Safety instructions risk and safety phrases The components of the Protino Ni IDA 150 1000 2000 packed columns Protino Ni IDA Resin and Protino Multi 96 Ni IDA kits do not contain hazardous contents 26 MACHEREY NAGEL 06 2006 Rev 03 Protino Ni IDA 5 Purification of polyhistidine tagged proteins from E coli under native conditions 5 1 Preparation of cleared lysates under native conditions Refer to Table 2 section 3 4 for detailed information on culture and buffer volume requirements Thaw the cell pellet from an E coli expression culture on ice if frozen Resuspend 1 g of pelleted wet cells in 2 5 ml LEW buffer for details see section 3 4 Pipette up and down or use stirring until complete resuspension without visible cell aggregates Perform this step on ice Add lysozyme to a final concentration of 1 mg ml Stir the solution on ice for 30 min Sonicate the suspension on ice according to the instructions provided by the manufacturer e g use 10 x 15 s bursts with a 15 s cooling period between each burst Carefully check samples appearance after sonication If the lysate is still viscous from incomplete fragmentation of DNA add 5 ug ml DNase and stir on ice for 15 min Centrifuge the crude lysate at 10 000 x g for 30 min at 4 C to remove cellular debris Carefully transfer the supernatant to a clean tube without disturbing the pellet
23. ffer compositions refer to section 3 6 2 e Protino Ni IDA Resin Purification under native conditions LEW buffer Elution buffer sodium phosphate sodium chloride imidazole For buffer compositions refer to section 3 6 1 Purification under denaturing conditions LEW buffer Denaturing Solubilization buffer Denaturing Elution buffer sodium phosphate sodium chloride urea and imidazole For buffer compositions refer to section 3 6 2 Protino Multi 96 Ni IDA Purification under native conditions Kits already contain buffer stock solutions that have to be prepared according to the instructions section 5 7 22 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 3 8 Protino Columns for gravity flow column chromato graphy using Protino Ni IDA Resin For column IMAC using Protino Ni IDA Resin we generally recommend gravity flow procedure For this MACHEREY NAGEL offers Protino Columns 14 ml and 35 ml Protino Columns are empty polypropylene columns with an inserted filter frit Separate frits for covering the column bed are also included Protino Columns are available with volume capacities of 14 ml and 35 ml see ordering information They can be used to retain up to 1 4 g and 3 5 g of Protino Ni IDA Resin respectively These maximum amounts of resin correspond to a protein binding capacity of 14 mg and 35 mg respectively for 6xHis GFPuv concentration 2 mg ml For detailed information
24. g cell 3 g cell 30 g cell pellet pellet pellet pellet resuspend in resuspend in resuspend in resuspend in 4 ml LEW 16 ml LEW 15 ml LEW 60 ml LEW ratio 1 5 ratio 1 2 ratio 1 5 ratio 1 2 4 ml of 16 ml of 15 ml of 60 ml of protein lysate protein lysate protein lysate protein lysate conc 5 mg ml conc 1 25 mg ml conc 5 mg ml conc 1 25 mg ml 18 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 3 5 Binding washing and elution In contrast to traditional IDA matrices Protino Ni IDA shows an optimized low density of IDA ligands which is created by a special manufacturing process This non saturating surface concentration of IDA eliminates non specific interactions of contaminating proteins with the adsorbent Since virtually no contaminating host proteins bind to Protino Ni IDA stringent washing procedures are generally not necessary Therefore LEW buffer which is used for lysis equilibration and washing does not contain any imidazole Bound polyhistidine tagged protein can competitively be eluted by adding imidazole The recommended Elution buffer contains 250 mM imidazole in order to recover even strong binding multimeric proteins with more than one polyhistidine tag also see buffer compositions section 3 6 However as shown in Figure 5 depending on the protein elution may be equally effective in the presence of much lower imidazole concentrations If for example the stabil
25. ice for 15 min Centrifuge the crude lysate at 10 000 x g for 30 min at 4 C to collect the inclusion bodies Discard supernatant Keep pellet on ice 2 Solubilization of inclusion bodies Resuspend the pellet in 10 ml LEW buffer per g Protino Ni IDA Resin to wash the inclusion bodies Centrifuge the suspension at 10 000 x g for 30 min at 4 C Discard supernatant MACHEREY NAGEL 06 2006 Rev 03 39 40 Protino Ni IDA Resuspend the pellet in 2 0 ml Denaturing Solubilization buffer per g Protino Ni IDA Resin to solubilize the inclusion bodies Homogenization or sonication may be necessary to resuspend the pellet Dissolve the inclusion bodies by stirring on ice for 60 min Centrifuge at 10 000 x g for 30 min at 20 C to remove any remaining insoluble material Carefully transfer the supernatant to a clean tube without disturbing the pellet If the supernatant is not clear centrifuge a second time or filter through a 0 45 um membrane e g celluloseacetate to avoid clogging of the IMAC column with insoluble material Save supernatant Proceed to section 6 2 or 6 3 MACHEREY NAGEL 06 2006 Rev 03 6 2 Protino Ni IDA Protino Ni IDA packed columns purification under denaturing conditions Protino Ni IDA packed columns Cell Extract Preparation Refer to section 6 1 For detailed information on culture and buffer volumes for cell extract preparation also see Table 2 section 3 4
26. in each 74919010 10 preps Protino Ni IDA 150 packed columns contains 40 mg of resin each 29 1199 90 SO Preps Protino Ni IDA 1000 packed columns contains 250 mg of resin each 759150 3 preps Protino Ni IDA 1000 packed columns contains 250 mg of resin each TASI GQ 39 SO preps Protino Ni IDA 2000 packed columns contains 500 mg of resin each proe SPIER Protino Ni IDA 2000 packed columns contains 500 mg of resin each TAOLINE e preps Protino Ni TED Resin 745200 5 5g Protino Ni TED Resin 745200 30 30g Protino Ni TED Resin 745200 120 120g Protino Ni TED Resin 745200 600 600 g Protino Ni TED 150 packed columns contains 40 mg of resin each PAS 100710 19 reps Protino Ni TED 150 packed columns contains 40 mg of resin each 749409 90 2O preps Protino Ni TED 1000 packed columns contains 250 mg of resin each TEILT Do Sepe Protino Ni TED 1000 packed columns 745110 50 EO pices contains 250 mg of resin each 46 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins Product Cat No Pack of Protino Ni TED 2000 packed columns contains 500 mg of resin each 1491209 9 peps Protino Ni TED 2000 packed columns contains 500 mg of resin each PAETE o preps Protino columns 14 ml 745250 10 10 columns Protino columns 35 ml 745255 10 10 columns NucleoBond rack small for Protino Ni TED IDA 150 packed columns ees NucleoBond rack large for Protino Ni TED IDA 1000 2000 pack
27. in in degassed deionized water Do not use a magnetic stirrer to avoid generating fine particles through excessive physical force Slowly pour the suspension into the column Avoid introducing air bubbles 1 g of Protino Ni IDA Resin will result in 2 ml bed volume The amount of resin required depends on the amount of polyhistidine tagged protein to be purified The binding capacity of Protino Ni IDA Resin varies from protein to protein See section 3 3 for general guidelines Allow the resin to settle Insert and adjust top adapter and connect the column to the chromatography system according to the manufacturer s instructions Avoid introducing air bubbles Column equilibration Equilibrate the column with 4 bed volumes of LEW buffer or until the baseline at 280 nm is stable Binding Apply the cleared lysate see section 5 1 to the column Washing Wash the column with 8 bed volumes of LEW buffer or until the baseline at 280 nm is stable Do not add imidazole to the LEW buffer MACHEREY NAGEL 06 2006 Rev 03 Protino Ni IDA Elution Elute the polyhistidine tagged protein with 5 10 bed volumes of Elution buffer using a step gradient When monitoring protein elution note that imidazole absorbs at 280 nm Note Depending on protein characteristics 9096 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of
28. ino Ni IDA products enable routine purification of recombinant polyhis tidine tagged proteins under native or denaturing conditions e Although designed for the purification of polyhistidine tagged proteins from E coli Protino Ni IDA products can also be used for the purification of polyhistidine tagged proteins from other expression systems including insect cells mammalian cells and yeast e The capacity of Protino Ni IDA see Table 1 was determined by using polyhistidine tagged green fluorescent protein 6xHis GFPuv 32 kDa expressed in E coli Capacities will vary for each His tagged protein 3 2 Purification under native and denaturing conditions This manual describes methods for the preparation of cell extracts from E coli and procedures for the purification of polyhistidine tagged recombinant proteins using Protino Ni IDA If recombinant proteins are expressed in E coli ideally the target proteins remain soluble in the cytoplasm However especially proteins that are highly expressed accumulate in insoluble aggregates which are called inclusion bodies For solubili zation 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 5 as well as insoluble proteins from inclusion bodies purification under denaturing conditions see section 6 In general for purification of polyhistidi
29. irectly correlate with the expression level and the cell density of the expression culture Therefore binding capacity will vary for each polyhistidine tagged protein and has to be determined for each expression experiment The binding behaviour of any polyhistidine tagged protein to Protino Ni IDA can be examined by calculating the amount of protein that is eluted as a function of the amount of protein that has been loaded see Figure 2 Please note that the resulting graph will vary in dependence on characteristics and concentration of the individual His tag protein The binding curve can be divided in three stages 1 Stage of maximum recovery At this stage the loaded protein is bound to the resin nearly quantitatively and can be eluted nearly quantitatively too the binding curve is almost linear see Figure 2 _ eluted His GFPuv 2 Stage of increasing yield decreasing recovery At this stage the binding curve becomes non linear and finally binding approaches saturation The protein yield increases with further increasing amount of loaded protein 3 Stage of maximum yield minimum recovery When loading excess protein the available binding sites of the resin are saturated The amount of eluted protein reaches a maximum The binding capacity for each individual protein can be defined as the yield at which the binding curve changes from the stage of maximum recovery to the stage of increasing yield decreasing recovery This point is an
30. ity Elution Elute the polyhistidine tagged protein in three fractions Add 3 x 3 bed volumes of Elution buffer and collect separately Allow the column to drain by gravity Note Depending on protein characteristics 9096 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 7 Storage reuse and regeneration of Protino Ni IDA Resin Storage After use wash resin with 10 bed volumes of LEW buffer and 10 bed volumes of deionized water Wash with 2 bed volumes of 2096 ethanol and store resin in 2096 ethanol at 4 C Reuse Depending on the nature of the sample Protino Ni IDA Resin can be reused 3 5 times Reuse should only be performed with identical polyhistidine tagged proteins to avoid possible cross contamination After the final elution step wash Protino Ni IDA Resin with the following solutions 5 bed volumes of 100 mM EDTA pH 8 5 bed volumes of deionized water 5 bed volumes of 100 mM NiSO 10 bed volumes of deionized water After equilibrating with LEW buffer the resin is ready for reuse Complete regeneration If a complete regeneration is mandatory wash resin with the following solutions 2 bed volumes of 6 M GuHCI 0 2 M acetic acid 5 bed volumes of deionized water 3 bed volumes of 2 9
31. ity or integrity of the target protein in 250 mM imidazole is a concern the concentration of imidazole in the eluent may readily be reduced Protino Ni IDA Ni IDA Agarose M CL Wash Elution M CL Wash Elution il 10 20 50 100 250 500 mM Imidazole 10 20 50 100 250 500 mM Imidazole KDa KDa 904 at 94 67 67 00 ESL 4 w 43 a s e ew e en 5 o 30 30 20 1 a 20 1 14 4 vm 14 4 Figure 5 Purification of polyhistidine tagged GFPuv using Protino Ni IDA and Ni IDA Agarose Recombinant GFPuv was expressed in E coli lysed loaded onto each gravity flow column and eluted by a stepwise imidazole gradient Eluted fractions were analyzed by SDS PAGE Pure polyhistidine tagged protein can be eluted from Protino Ni IDA left panel at much lower imidazole concentrations than from Ni IDA Agarose Ped panel In addition Ni IDA Agarose releases contaminating proteins from 10 mM to 100 mM imidazole M Marker proteins CL Cleared lysate MACHEREY NAGEL 06 2006 Rev 03 19 Purification of Polyhistidine Tagged Proteins 3 6 Buffer compositions 3 6 1 Buffers for purification under native conditions Protino Ni IDA 150 1000 2000 packed columns kits and Protino Multi 96 Ni IDA kits contain LEW Elution buffer stock solutions that have to be diluted according to the instructions given in the individual protocol see sections 5 2 and 5 7 Protino Ni IDA Resin
32. ive conditions using lysozyme together with sonication After centrifugation the polyhistidine tagged protein is extracted and solubilized from the pellet by using a denaturant 8 M urea The extract obtained is clarified by centrifugation and applied to Protino Ni IDA packed columns or Protino Ni IDA Resin under denaturing conditions Purification of polyhistidine tagged proteins under denaturing conditions is similar to purification under native conditions except that the cell extract and buffers loaded on the column contain 8 M urea For buffer compositions see section 3 6 6 1 Cell extract preparation under denaturing conditions 1 Isolation of inclusion bodies Refer to Table 2 section 3 4 for detailed information on culture and buffer volume requirements Thaw the cell pellet from an E coli expression culture on ice if frozen Resuspend 1 g of pelleted wet cells in 5 ml LEW buffer without denaturant on ice also see section 3 4 Pipette up and down or use stirring until complete resuspension without visible cell aggregates Add lysozyme to a final concentration of 1 mg ml Stir the solution on ice for 30 min Sonicate the suspension on ice according to the instructions provided by the manufacturer e g use 10 x 15 s bursts with a 15 s cooling period between each burst Carefully check samples appearance after sonication If the lysate is still viscous from incomplete fragmentation of DNA add 5 pg ml DNase and stir on
33. kits do not contain any buffers Prepare LEW buffer and Elution buffer according to the instructions given in this section Note that lysis buffer equilibration buffer and washing buffer are the same Note Do not include any imidazole in the Lysis Equilibration Wash buffer since most proteins do not bind to the resin in the presence of even low imidazole concentration Lysis Equilibration Wash buffer LEW buffer 1 liter e 50mM NaH PO 7 8 g NaH 2PO x 2 H2O MW 156 01 g mol e 300 mM NaCl 17 5 g NaCl MW 58 44 g mol Adjust pH to 8 0 using NaOH Elution buffer 1 liter e 50 mM NaH PO 7 8 g NaH 2PO x 2 H2O MW 156 01 g mol e 300 mM NaCl 17 5 g NaCl MW 58 44 g mol e 250 mM imidazole 17 0 g imidazole MW 68 08 g mol Adjust pH to 8 0 using NaOH 20 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 3 6 2 Buffers for purification under denaturing conditions Protino Ni IDA 150 1000 2000 packed columns kits contain stock solutions of LEW buffer and Elution buffer for purification under native conditions For purification under denaturing conditions prepare Denaturing Solubilization buffer and Denaturing Elution buffer according to the instruction given in this section Note that additional volumes of LEW buffer have to be prepared as well Protino Ni IDA Resin kits do not contain any buffers Prepare LEW buffer Denaturing Solubilization buffer and Denaturing
34. ne tagged proteins the bacterial cells are disrupted using lysozyme in combination with sonication After centrifugation soluble target protein is found in the supernatant while inclusion bodies remain in the pellet The clear supernatant can directly be subjected to further purification using Protino Ni IDA packed columns or Protino Ni IDA Resin under native conditions see section 5 In case of massive formation of inclusion bodies the target protein is extracted from the pellet using a denaturant 8 M urea and further purified using protocols for the purification under denaturing conditions see section 6 If the distribution of the recombinant protein is unknown it is recommended to perform SDS PAGE analysis using the crude cell extract prior to centrifugation and the clear supernatant after centrifugation While the crude cell extracts will contain both soluble and insoluble target protein only soluble target protein is found in the supernatant MACHEREY NAGEL 06 2006 Rev 03 11 Purification of Polyhistidine Tagged Proteins 3 3 Binding capacity of Protino Ni IDA 3 3 1 General information The binding capacity of Protino Ni IDA strongly depends on the characteristics of the polyhistidine tagged protein e g amino acid composition molecular weight 3 D structure oligomerization properties etc Furthermore the absolute yield also depends on the total amount and concentration of the target protein in the sample which in turn d
35. o ensure appropriate flow rates 1 Column preparation Transfer the appropriate amount of Protino Ni IDA Resin to an empty Protino Column To achieve tight packing gently tap the column on a hard surface until the bed height remains constant Place a separate filter frit on top of the column bed by using a lab pen Gently tap on the frit to ensure that there is no gap between column bed and filter frit 1 g of Protino Ni IDA Resin will result in 2 ml bed volume The amount of resin required depends on the amount of polyhistidine tagged protein to be purified The binding capacity of Protino Ni IDA Resin varies from protein to protein See section 3 3 for general guidelines 2 Column equilibration Equilibrate the column with 4 bed volumes of LEW buffer Allow the column to drain by gravity 3 Binding Add the supernatant cleared lysate see section 5 1 to the pre equilibrated column and allow the column to drain by gravity Apply at least 1 5 bed volumes of sample 4 Washing Wash the column twice with 4 bed volumes of LEW buffer Allow the column to drain by gravity 5 Elution Elute the polyhistidine tagged protein in three fractions Add 3 x 3 bed volumes of Elution buffer and collect separately Allow the column to drain by gravity Note Depending on protein characteristics 9096 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s
36. of Elution buffer and collect separately Allow the column to drain by gravity Note Depending on protein characteristics 9096 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein 32 MACHEREY NAGEL 06 2006 Rev 03 Protino Ni IDA 5 5 Protino Ni IDA Resin batch purification protocol under native conditions Although we recommend gravity flow procedure polyhistidine tagged proteins may be purified by the following batch protocol 1 Batch binding Add the appropriate amount of Protino Ni IDA Resin directly to the cleared lysate see section 5 1 filled in a centrifugation tube Close the tube and mix the suspension gently e g on an orbital shaker for 5 15 min The amount of resin required depends on the amount of polyhistidine tagged protein to be purified The binding capacity of Protino Ni IDA Resin varies from protein to protein See section 3 3 for general guidelines 1 g of Protino Ni IDA Resin will result in 2 ml bed volume The length of time required for optimal binding will vary from protein to protein Sediment the resin by gravity or centrifugation at 500 x g for 1 min Carefully decant the supernatant and discard it 2 Washing Add 4 bed volumes of LEW buffer and mix for 5 min Sediment the resin by gravity or centrifugation at 500 x g for 1 min Carefully dec
37. olumn chromatography under native conditions 34 5 7 Protino Multi 96 Ni IDA purification under native conditions 36 6 Purification of polyhistidine tagged proteins from E coli under denaturing conditions 39 6 1 Cell extract preparation under denaturing conditions 39 MACHEREY NAGEL 06 2006 Rev 03 3 Purification of Polyhistidine Tagged Proteins 6 2 Protino Ni IDA packed columns purification under denaturing conditions 41 6 3 Protino Ni IDA Resin gravity flow column chromatography under denaturing conditions 42 7 Storage reuse and regeneration of Protino Ni IDA Resin 43 8 Appendix 44 8 1 Troubleshooting 44 8 2 Ordering information 46 8 3 Product use restriction warranty 47 4 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins 1 Contents Protino Ni IDA 150 packed columns 10 preps 50 preps Cat No 745150 10 745150 50 Protino Ni IDA 150 packed columns 10 50 8x LEW buffer 5 ml 25 ml 4x Elution buffer 5 ml 20 ml Protocol 1 1 Protino Ni IDA 1000 packed columns 5 preps 50 preps Cat No 745160 5 745160 50 Protino Ni IDA 1000 packed columns 5 50 8x LEW buffer 30 ml 2x 70 ml 4x Elution buffer 25 ml 115 ml Plastic washer 5 8 Protocol 1 1 Protino Ni IDA 2000 packed columns 5 preps 25 preps Cat No 745170 5 745170 25 Protino Ni IDA 2000 packed columns 5 25 8x LEW buffer 30 ml 2x70ml 4x Elution buffer 25 ml 115 ml Plastic washer 5 8 Protocol 1 1
38. olumns are designed to fit into most 15 ml conical centrifuge tubes e g BD Falcon Cat No 352097 for convenient fraction collection Binding Add the cleared lysate see section 5 1 to the pre equilibrated column and allow the column to drain by gravity Washing Wash the column with 1x LEW Buffer Allow the column to drain by gravity Elution Elute the polyhistidine tagged protein in a new collecting tube by adding 1x Elution Buffer Allow the column to drain by gravity TT ixiSm MEE Note Depending on protein characteristics 90 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 Protino Ni IDA 5 3 Protino Ni IDA Resin gravity flow column chromatography under native conditions For column IMAC using Protino Ni IDA Resin we generally recommend gravity flow procedure This protocol describes gravity flow column chromatography using Protino Ni IDA Resin and Protino Columns see section 3 8 Prepacked columns filled with 40 mg 250 mg or 500 mg Protino Ni IDA Resin may readily be used Protino Ni IDA 150 1000 or 2000 packed columns see section 5 2 and ordering information Note When using other types of chromatography columns please note that the pore size of the filter frit should be around 50 um t
39. optimal compromise between protein load and recovery and will vary for each individual protein 3 3 2 Binding capacity The binding capacity of Protino Ni IDA is exemplified using the green fluorescent protein 6xHis GFPuv 32 kDa at a concentration of 2 mg ml Please note that different recombinant proteins may show a different binding behaviour Figure 2 shows a plot of the amount of eluted 6xHis GFPuv against the amount of loaded 6xHis GFPuv The binding curve can be divided in three stages 1 Stage of maximum recovery 20 mg 6xHis GFPuv load g resin 2 Stage of increasing yield decreasing recovery gt 20 mg 6xHis GFPuv load g resin 3 Stage of maximum yield minimum recovery gt 75 mg 6xHis GFPuv load g resin 12 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins Under the above mentioned conditions the binding capacity of Protino Ni IDA for 6xHis GFPuv is approximately 20 mg protein per g of resin see arrow Figure 2 At this point the protein recovery is gt 80 Consequently the following amounts of 6xHis GFPuv have to be loaded For optimal recovery load 20 mg protein per 1 g of Protino Ni IDA Resin for maximum yield load 75 mg protein per 1 g of Protino Ni IDA Resin 50 100 gt as gt A 49 4 a eo 8 Q O o DX Q I 30 4 60 9 J O 2 D O 10 4 20 eluted 6xHis GFPuv O recovery 0 10 20 30 40 50 60 70 80 loaded 6xHis GFPuv mg g
40. otino Ni IDA Resin and Protino Columns see section 3 8 Prepacked columns filled with 40 mg 250 mg or 500 mg Protino Ni IDA Resin readily be used Protino Ni IDA 150 1000 or 2000 packed columns see section 6 2 and ordering information Note When using other types of chromatography columns please note that the pore size of the filter frit should be around 50 um to ensure appropriate flow rates 42 Column preparation Transfer the appropriate amount of Protino Ni IDA Resin to an empty Protino Column To achieve tight packing gently tap the column on a hard surface until the bed height remains constant Place a separate filter frit on top of the column bed by using a lab pen Gently tap on the frit to ensure that there is no gap between column bed and filter frit 1 g of Protino Ni IDA Resin will result in 2 ml bed volume The amount of resin required depends on the amount of polyhistidine tagged protein to be purified The binding capacity of Protino Ni IDA Resin varies from protein to protein See section 3 3 for general guidelines Column equilibration Equilibrate the column with 4 bed volumes of Denaturing Solubilization buffer Allow the column to drain by gravity Binding Add the supernatant cleared lysate see section 6 1 to the pre equilibrated column and allow the column to drain by gravity Washing Wash the column with 8 bed volumes of Denaturing Solubilization buffer Allow the column to drain by grav
41. purification procedure First time users are strongly advised to read this user manual The Protino Ni packed columns protocols in this manual are organized as follows The culture volumes and volumes of the respective buffers used for a particular column size are highlighted Each procedural step is arranged like the following example taken from section 5 2 2 Protino Ni IDA packed columns 150 1000 EUM 3 Column equilibration Equilibrate Protino Ni packed columns with 1x LEW Buffer Allow the column to drain by gravity 2m ETH Protino Ni 150 1000 packed columns are designed to fit into most 15 ml conical centrifuge tubes e g BD Falcon Cat No 352097 for convenient fraction collection 8 MACHEREY NAGEL 06 2006 Rev 03 Purification of Polyhistidine Tagged Proteins For example if you are using Protino Ni IDA 150 packed column you are requested to refer to the white boxes These boxes indicate the volume of bacterial culture or buffer to be used The respective buffer is highlighted in bold type within the instruction Referring to the a m example there has to be used 320 ul of LEW buffer for column equilibration when using Protino Ni IDA 150 packed columns MACHEREY NAGEL 06 2006 Rev 03 9 Purification of Polyhistidine Tagged Proteins 3 Product description 3 1 Specifications Table 1 Specifications Protino Ni IDA packed columns 150 1000 2000 Protein capacity
42. the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 35 Protino Ni IDA 5 7 Protino Multi 96 Ni IDA purification under native 36 conditions Cultivate and harvest cells E coli cells 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 2000 x g for 15 min at 4 C Store cell pellets at 20 C or 70 C for at least 1 h Preparation of working solutions Prepare 1x LEW Lysis Equilibration Wash buffer and 1x Elution buffer by diluting the supplied stock solutions Note If precipitate is observed in the stock solutions warm and shake them to dissolve precipitate prior to diluting the buffers Mix 50 ml of 8x LEW buffer with 350 ml of deionized water to get a final volume of 400 ml 1x LEW buffer sufficient for 96 preps Mix 25 ml of 4x Elution buffer with 75 ml of deionized water to get a final volume of 100 ml of 1x elution buffer sufficient for 96 preps Cell extract preparation 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 Cell extracts from 5 ml E coli expression cultur
43. ts Sets of 1 rack 12 strips with 8 tubes each MACHEREY NAGEL 06 2006 Rev 03 47 Purification of Polyhistidine Tagged Proteins MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER 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
44. umns purification under Protino Ni IDA native conditions Note Experienced users may refer to the protocol at a glance section 5 2 1 Protino Ni IDA packed columns 1000 1 Cultivate and harvest cells Harvest cells from an E coli expression culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C Remove supernatant Store cell pellet at 20 C if not processed immediately 2 Preparation of working solutions Prepare 1x LEW Lysis Equilibration Wash buffer and 1x Elution buffer by diluting the supplied stock solutions Note If precipitate is observed in the stock solutions warm and shake them to dissolve precipitate prior to diluting the buffers Mix 8x LEW buffer 1 5 ml with deionized water 10 5 ml 17 5 ml to get a final volume of 1x LEW buffer sufficient for one column run 2 4 mi 12 ml 20mi Mix 4x Elution buffer with deionized water 0 750 m ismi ET to get a final volume of 1x elution buffer sufficient for one column run 6 ml MACHEREY NAGEL 06 2006 Rev 03 29 Protino Ni IDA Protino Ni IDA packed columns 1000 EN 30 Cell Extract Preparation Refer to section 5 1 For detailled information on culture and buffer volumes for cell extract preparation also see Table 2 section 3 4 Column equilibration Equilibrate Protino Ni IDA packed columns with 1x LEW Buffer Allow the column to drain by gravity Protino Ni IDA 150 1000 packed c
45. ven low imidazole concentrations Washing Wash each well twice by adding 800 pl of 1x LEW buffer Allow the wells to drain by gravity Note Stringent washing procedures by using buffers with low imidazole concentrations are generally not necessary Therefore LEW buffer does not contain any imidazole 1 Benzonase is a registered trademark of Merck KGaA Darmstadt Germany MACHEREY NAGEL 06 2006 Rev 03 37 Protino Ni IDA 38 Elution Elute the polyhistidine tagged protein in a suitable collecting plate e g MN Tube Strips see ordering information by adding 3 x 250 ul of 1x Elution buffer Allow the wells to drain by gravity Note To increase the concentration of the polyhistidine tagged protein in the final eluate add 80 yl of Elution buffer and discard the flow through this flow through mainly contains LEW buffer from the washing steps Afterwards elute the protein by adding 3 x 250 yl of Elution Buffer Note Depending on protein characteristics 90 of the eluted protein can be found in the first elution fraction Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein MACHEREY NAGEL 06 2006 Rev 03 Protino Ni IDA 6 Purification of polyhistidine tagged proteins from E coli under denaturing conditions We recommend this protocol if expression leads to the formation of inclusion bodies Cells are disrupted under nat
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