MoBiTec Ni-IDA Columns for His-Tag Purification
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1. Secretory proteins contain a signal peptide that addresses them for the export into the periplasmic space e g E coli or into the culture medium e g Bacillus spec Please consider that in case of secretory proteins the polyhistidine tag must be located C terminal of the protein Otherwise the tag will be cleaved of during signal peptide processing 5 4 Purification under native and denaturing conditions The decision whether to purify polyhistidine tagged proteins under native or denaturing conditions depends on diverse considerations protein location and solubility the accessibility of the polyhistidine tag and whether biological activity must be retained Depending on the expression system and the host the recombinant proteins will accumulate in the cytoplasm or will be secreted into the periplasmic space or out into the culture medium Secreted protein is in most cases correctly folded and soluble Intracellularly accumulated recombinant protein remains either in a soluble form or aggregates as insoluble misfolded protein in inclusion bodies In case of soluble protein purified from cytoplasm periplasm or supernatant with good accessibility of the polyhistidine tag the purification can be done under native conditions see protocol 6 3 1 Preparation of cleared lysates under native conditions Native conditions may also be used if co purification of associated proteins is desired In case of inclusion body formation the poly2. buffer e Store supernatant on ice e Proceed with the protocol 6 3 1 Preparation of cleared lysates under native conditions MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 8 6 3 Purification of Intracellular polyhistidine tagged proteins under native or denaturing conditions For purification of intracellular polyhistidine tagged proteins under native conditions a considerable portion of the protein should be present in a soluble form If so please follow the protocol 6 3 1 Preparation of cleared lysates under native conditions High levels of recombinant protein expression might lead to the formation of insoluble aggregates in E coli these are known as inclusion bodies During preparation of intracellular recombinant protein cells are disrupted and cell pellets are separated from the soluble fraction by centrifugation In contrast to soluble protein that remains in the supernatant the inclusion bodies will sediment with the cell debris and the polyhistidine tagged protein has to be extracted from cell pellet using urea as denaturant 8 M urea completely solubilizes the inclusion bodies and 6xHis tagged proteins Under these denaturing conditions the 6xHis tag on a protein will be fully exposed so that binding to the Ni IDA matrix is enabled If most of the 6xHis tagged protein is localized within inclusion bodies please follow
3. denaturing conditions 10 6 6 Storage Reuse and Regeneration ss 10 6 7 Compatibility Of reagents ee 12 6 8 TOUBISSROO NOEL Sn Re nana re 13 7 Order Information Shipping and Storage ne 14 8 Contact and SUpport ee d care crane 14 MoBiTec GmbH Germany Phone 49 551 707220 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 3 1 Features Excellent tool for routine purification of recombinant polyhistidine tagged proteins under native and denaturating conditions starting from diverse expression systems e g E coli yeast insect and mammalian cells Maximal binding capacity 90 mg protein per column Protein recovery rate gt 80 Improved target specificity by optimized silica based Ni IDA matrix Imidazol free loading and washing buffer Columns are long term storable when kept dry 2 Introduction MoBiTec Ni IDA Columns with silica based resin provide a fast and convenient routine tool for purification of recombinant polyhistidine tagged proteins by gravity flow The form stable silica matrix is precharged with Ni ions and allows purification on the principle of Immobilized Metal lon Affinity Chromatography IMAC Binding of proteins is based on the interaction between the polyhistidine tag of the recombinant protein and immobilized Ni ions The chelating group of the Ni IDA resin is based on IDA iminodiacetic acid which enables stro
4. proteins to avoid possible cross contamination After the final elution step wash the column with the following solutions 10 ml 100 mM EDTA pH 8 10 ml deionized water 10 ml 100 mM NiSO4 10 ml deionized water After equilibrating with LEW buffer or Denaturing Solubilization Buffer the column is ready for reuse oe Sh MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 11 6 For storage overnight we recommend to store the column with LEW buffer at 4 C Otherwise follow the protocol for long term storage below 6 6 2 Long term storage After performing the reuse protocol wash column with 20 ml LEW buffer 20 ml deionized water 4 ml 20 ethanol Store column with 20 ethanol at 4 C After equilibrating with 20 ml LEW buffer the column is ready for reuse eid I 6 6 3 Complete regeneration If a complete regeneration is mandatory wash the column with the following solutions 1 4 ml 6 M GuHCl 0 2 M acetic acid 10 ml deionized water 6 ml 2 SDS 10 ml deionized water 10 ml 100 ethanol 10 ml deionized water 10 ml 100 mM EDTA pH 8 10 ml deionized water 10 ml 100 mM NiSO 20 ml deionized water After equilibrating with 20 ml LEW buffer the column is ready for reuse 2 3 4 5 6 7 8 9 1 1 MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com O
5. recommended but up to causing a decrease in capacity 0 5 in samples has been used successfully in some cases Sodium chloride Prevents ionic interactions and therefore unspecific binding up to 2 M can be used at least 0 3 M should be used 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 Triton Tween Removes background proteins Up to 2 can be used Urea Solubilizes proteins Use 8 M urea for purification under denaturing conditions Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH Germany MoBiTec GmbH 2015 6 8 Troubleshooting Problem Caused by Suggestions Sample does not enter the column bed Sample lysate contains insoluble material If sample is not clear use centrifugation of filtration 0 45 um membrane to avoid clogging of the column 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 Protein does not bind to the resin Vector const
6. 0722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 7 Order Information Shipping and Storage Order Product Amount PR HTK004 MoBiTec Ni IDA Columns 4 columns PR HTK010 MoBiTec Ni IDA Columns 10 columns shipped at RT store columns at RT 8 Contact and Support MoBiTec GmbH Lotzestrasse 22a D 37083 Goettingen Germany Customer Service General inquiries amp orders Technical Service Product information phone 49 0 551 707 22 0 phone 49 0 551 707 22 70 fax 49 0 551 707 22 22 fax 49 0 551 707 22 77 e mail order mobitec com e mail info mobitec com MoBiTec in your area Find your local distributor at www mobitec com MoBiTec GmbH Germany Phone 49 551 707220 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com
7. MoBiTec GmbH 2015 Page 12 6 7 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 gt 8 4 since silica dissolves in solutions of high pH MoBiTec GmbH Germany Reagent Effect Comment B mercaptoethanol Prevents formation of disulfide bonds can reduce Ni ions at higher concentrations Up to 50 mM in samples has been successfully used in some cases DTT DTE Can reduce Ni ions at higher Up to 10 mM in samples has concentrations been successfully used in some cases EDTA Coordinates with Ni ions Not recommended but up to causing a decrease in capacity 1 mMin samples has been used at higher concentrations successfully in some cases Ethanol Prevents hydrophobic Up to 20 can be used ethanol interactions between proteins may precipitate proteins causing low flow rates and column clogging Glutathione reduced Can reduce Ni ions at higher concentrations Up to 30 mM in samples has been successfully used in some cases Glycerol Prevents hydrophobic Up to 50 can be used interactions between proteins GuHCl Solubilizes proteins Up to 6 M can be used Imidazole Binds to immobilized Ni ions Imidazole should not be included and competes with the in LEW buffer polyhistidine tagged proteins SDS Interacts with Ni ions Not
8. MoBiTec Ni IDA Columns TE e Mo Bi Tec MoBiTec GmbH 2015 Page 2 Contents T Feat re Siini a E tea ma nn 3 2 VELOC OU COR esene ER ERA RENE EEE RER RE PEER 3 3 Product Contents siscesissiccstnciatcndsssasscdncvatsadcdsncsieceadeeadduancsdudubsdedistanduaaduasenbesennedaadnaaaue 4 3 1 Kit COMponents nn nee ee ee 4 3 2 IS and EXPY seisoene ne deu 4 3 3 Equipment and materials to be supplied by user 4 3 4 Buffer COMPOSITIONS ne ene cttana tendon tee Rene n tre nn de Reed einen eue 4 4 Terms and Conditions ii sssisst snemencnesstelsssdenatsests son rec aaia ianei tiss eceatianssnss 5 5 TECHNICAL Information au on nn ananas anina ranana eee ninad anasinin E inai 5 Oe Protein DINGING RS A E E 5 5 2 BINGING CAPACI a en ee nee ne et een tn idea eee ed 6 5 3 Purification of secretory proteins 2505 immo mere meenes eee aschenuse 6 5 4 Purification under native and denaturing conditions 6 SR en aa aaa ee aaa ee eerie 7 6 PROLOCONS aE E E T T E E E 7 6 1 Purification of secreted polyhistidine tagged proteins from the supernatant 7 6 2 Purification of periplasmic polyhistidine tagged proteins 7 6 3 Purification of intracellular polyhistidine tagged proteins under native or denaturing COMOMIDING 5 2 nano lee di etais 8 6 4 Ni IDA chromatography under native conditions 9 6 5 Ni IDA chromatography under
9. crose Buffer Required for the following protocol 6 2 Purification of periplasmic polyhistidine tagged proteins 30 mM Tris HCl e 20 sucrose Adjust pH to 8 0 4 Terms and Conditions For research use only NOT recommended or intended for diagnosis of disease in humans or animals Do NOT USE internally or externally in humans or animals All chemicals should be considered as potentially hazardous Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products Suitable protective clothing such as laboratory overalls safety glasses and gloves should be worn Care should be taken to avoid contact with skin or eyes if contact should occur wash immediately with water See Material Safety Data Sheet s Product warranty is limited to our liability to replacement of this product All other warranties expressed or implied including but not limited to any implied warranties of merchantability or fitness for a particular purpose are excluded and do not apply We shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product 5 Technical Information 5 1 Protein binding Proteins containing one or more polyhistidine affinity tags located at either the amino or carboxyl terminus of the protein can bind to the Ni IDA matrix with very high affinity Even when
10. eins cells and supernatant will be separated by centrifugation 15 min 4500 x g to 6000 x g 4 C The clear supernatant can be directly subjected to MoBiTec Ni IDA columns Please follow the protocol 6 3 1 Preparation of cleared lysates under native conditions 6 2 Purification of periplasmic polyhistidine tagged proteins Periplasmic proteins can be separated from cytoplasmic proteins by osmotic shock preparation see protocol below The obtained osmotic shock fluid can then be subjected to MoBiTec Ni IDA columns e Harvest the cells from expression culture by centrifugation 15 min 4500 x g to 6000 x g 4 C e Resuspend cell pellet in Sucrose Buffer at 80 ml per gram wet weight e Keep the cells on ice and add 500 mM EDTA solution dropwise to 1 mM final concentration e Incubate the cells on ice for 5 10 min with gentle agitation e Centrifuge the cell suspension at 8000 x g for 20 min at 4 C e Remove supernatant completely and resuspend the pellet in the same volume of ice cold 5 mM MgSO solution e Shake or stir for 10 min in an ice bath e Centrifuge at 8000 x g for 20 min at 4 C e Carefully transfer the supernatant containing the periplasmic proteins to a clean tube without disturbing the pellet e f the supernatant is not clear centrifuge a second time or filter through a 0 45 um membrane to avoid clogging of the Ni IDA column with insoluble material e Dialyze the supernatant extensively against LEW
11. g on culture volume e Sonicator e Lysozyme e Optional Phenylmethylsulfonyl fluoride PMSF DNase 0 45 um membrane filter e 500 mM EDTA and 5 mM MgSO only for preparation of periplasmic proteins e Buffers according to protocol composition of all buffers see 3 4 3 4 Buffer compositions Lysis Equilibration Wash Buffer LEW Buffer Required for the following protocol 6 3 1 Preparation of cleared lysates under native conditions 6 3 2 Preparation of cleared lysates under denaturing conditions 50 mM NaH2PO4 300 mM NaCl e adjust pH to 8 0 using NaOH Elution Buffer Required for the following protocol 6 3 1 Preparation of cleared lysates under native conditions 50 mM NaH2PO4 300 mM NaCl 250 mM imidazole adjust pH to 8 0 using NaOH Denaturing Solubilization Buffer Required for the following protocol 6 3 2 Preparation of cleared lysates under denaturing conditions Please prepare shortly before use 50 mM NaH2PO4 300 mM NaCl MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 5 e 8Murea e adjust pH to 8 0 using NaOH Denaturing Elution Buffer Required for the following protocol 6 3 2 Preparation of cleared lysates under denaturing conditions Please prepare shortly before use 50 mM NaH2PO4 e 300 mM NaCl e 8Murea e 250 mM imidazole e adjust to pH to 8 0 using NaOH Su
12. histidine tagged protein has to be extracted from cell pellet using urea as denaturant see protocol 6 3 2 Preparation of cleared lysates under denaturing conditions Denaturing conditions can also be an option for improving the accessibility of the polyhistidine tag Tabel 1 How to find the proper purification conditions please choose Native conditions if Denaturing conditions if protein is soluble protein aggregates e g inclusion bodies polyhistidine tag is good accessible the polyhistidine tag is hardly accessible co purification of associated proteins desired MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 7 5 5 Culture size The recommended culture size volume complies with the concentration of the polyhistidine tagged protein in the culture The latter typically varies from lt 10 mg l up to 100 mg l depending on cell density and expression level Table 2 Culture size guide Expression Recommended Recommended Estimated amount of poly level culture volume pellet wet weight histidine tagged protein in E coli sample High 100 mg l 0 2 1 0 8 4 g 20 100 mg Low 10 mg l 2 10 8 40 g 20 100 mg 6 Protocols 6 1 Purification of secreted polyhistidine tagged proteins from the supernatant e g B subtilis For preparation of secreted polyhistidine tagged prot
13. in total 18 ml Elution Buffer divided in a suitable number of fractions and collect separately Allow the column to drain by gravity Note Commonly 90 of the eluted protein can be found within the first 6 ml of elution Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein 6 5 Ni IDA chromatography under denaturing conditions Column Equilibration Equilibrate the column with 8 ml of Denaturing Solubilization Buffer Allow the column to drain by gravity Binding Add the cleared lysate at least 3 ml to the pre equilibrated column and allow the column to drain by gravity Washing Wash the column twice with 16 ml Denaturing Solubilization Buffer Allow the column to drain by gravity Elution Elute the polyhistidine tagged protein in 3 6 fractions Add in total 18 ml Denaturing Elution Buffer divided in a suitable number of fractions and collect separately Allow the column to drain by gravity Note Commonly 90 of the eluted protein can be found within the first 6 ml of elution Use protein assay and or SDS PAGE analysis to determine which fraction s contain s the majority of the polyhistidine tagged protein 6 6 Storage reuse and regeneration 6 6 1 Reuse and short term storage Depending on the nature of the sample MoBiTec Ni IDA Columns can be reused 3 5 times Reuse should only be performed with identical polyhistidine tagged
14. ng and efficient binding of target protein onto the IMAC matrix In contrast to traditional IDA matrices MoBiTec Ni IDA is an optimized matrix with low density of IDA ligands This non saturating surface concentration of IDA eliminates almost all non specific interactions of contaminating host proteins with the adsorbent As a result MoBiTec Ni IDA provides higher target protein purity IDA is a tridentate 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 Fig 1 HO Silica Bead Fig 1 Silica based Ni IDA matrix Schematic drawing of IDA in complex with Ni MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 4 3 Product Contents 3 1 Kit components Order Product Components included PR HTK004 MoBiTec Ni IDA Columns 4 columns with matrix dry prepacked user manual PR HTK010 MoBiTec Ni IDA Columns 10 columns with matrix dry prepacked user manual 3 2 Storage and expiry MoBiTec Ni IDA Columns are storable at room temperature for at least 1 year 3 3 Equipment and materials to be supplied by user e Microliter pipettes e Appropriate centrifuge 2 10000 x g and tubes for collection and centrifugation size is dependin
15. otein remains in the supernatant Clear supernatant can be directly subjected to MoBiTec Ni IDA Columns follow the protocol 6 4 Ni IDA chromatography under native conditions In case of inclusion body formation the 6xHis tagged protein has to be extracted from cell pellet using urea as denaturant follow the protocol below Isolation of inclusion bodies e Harvest the cells from expression culture by centrifugation 15 min 4500 xg to 6000 x g 4 C e Remove supernatant and store pellet at 20 C or proceed immediately Please perform all steps on ice e Thoroughly resuspend the pellet in LEW buffer 1 25 5 mg polyhistidine tagged protein 1 ml LEW see also table 3 by pipetting up and down or vortexing until complete resuspension is reached no cell aggregates visible anymore Add lysozyme to a final concentration of 1 mg ml and optional protease inhibitor e g 0 1 mM PMSF final concentration Stir the solution on ice for 30 min e Sonicate the suspension on ice according to the instructions provided by the manufacturer e g 10 x 15 sec burst with 20 sec rest amplitude of 20 e Carefully check sample appearance after sonication If the lysate is still viscous from incomplete fragmentation of DNA add 5 ug ml DNase and stir on ice for additional 15 min e Centrifuge the crude lysate at 10000 x g for 30 min at 4 C to collect inclusion bodies Discard the supernatant and keep the pellet on ice Solubilization of inclu
16. ruct is not correct Check if gene of interest and tag has been cloned in frame Binding conditions are incorrect Check composition of buffers and verify pH 7 8 Ensure that there is no chelating or strong reducing reagent or imidazole present Protein elutes with wash buffer Buffer compositions are incorrect Check composition of buffers and verify pH 7 8 Ensure that there is no chelating or strong reducing reagent or imidazole present Protein does not elute from column Elution conditions are too mild Increase concentration of imidazole Contamination of other proteins within the eluate Insufficient washing Use larger volumes for washing Binding and washing conditions are too mild Add small amounts of imidazole 1 10 mM Take care that the imidazole concentration remains low enough to enable binding of the polyhistidine tagged proteins Contaminating proteins and the polyhistidine tagged protein are connected via disulfide bonds Add up to 30 mM B mercapto ethanol to reduce disulfide bonds Contaminating proteins are degradation products of polyhistidine tagged protein Perform cell lysis at 4 C Include protease inhibitor Expression is too low this leads to increased binding of contaminating proteins Increase expression level Increase amount of culture volume or cell pellet weight Phone 49 551 70722 0 Fax 49 551 7
17. sion bodies e Resuspend the pellet in 10 ml LEW buffer to wash the inclusion bodies Centrifuge the suspension at 10000 x g for 30 min at 4 C and discard the supernatant e Resuspend the pellet in 2ml Denaturating Solubilization Buffer to solubilize the inclusion bodies For complete solubilization it may be necessary to vortex or sonicate the solution Stir the suspension for further 60 min on ice e Centrifuge at 10000 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 e f the supernatant is not clear centrifuge a second time or filter through a 0 45 um membrane to avoid clogging of the MoBiTec Ni IDA column with insoluble material e Store supernatant at 4 C Proceed with chapter 6 5 Ni IDA chromatography under denaturing conditions 6 4 Ni IDA chromatography under native conditions Column equilibration Equilibrate the column with 8 ml LEW buffer Allow the column to drain by gravity MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 10 Binding Add the cleared lysate at least 3 ml to the pre equilibrated column and allow the column to drain by gravity Washing Wash the column three times with 8 ml LEW buffer 3 x 8 ml Allow the column to drain by gravity Elution Elute the polyhistidine tagged protein in 3 6 fractions Add
18. ter through a 0 45 um membrane to avoid clogging of the Ni IDA column with insoluble material e Store supernatant on ice Proceed with chapter 6 4 Ni IDA chromatography under native conditions Table 3 LEW buffer volume guide Expression Recommended Recommended Ratio pellet Volume of level culture volume pellet wet weight weight LEW LEW for re volume suspension High 100 mg l 200 ml minimum 0 8 g minimum 1 5 4 ml 1 maximum 4 g maximum 1 5 20 ml Low 10 mg l 21 minimum 8 g minimum 1 2 16 ml 10 maximum 40 g maximum 1 2 80 ml MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 Page 9 6 3 2 Preparation of cleared lysates under denaturing conditions E coli High levels of expression of recombinant proteins in a variety of expression systems can lead to the formation of insoluble aggregates in E coli these are known as inclusion bodies Strong denaturants such as 8 M urea completely solubilize inclusion bodies and 6xHis tagged proteins Under these conditions the 6xHis tag on a protein will be fully exposed so that binding to the Ni IDA matrix may be improved For preparation of intracellular recombinant proteins cells are disrupted and cell pellets are separated from the soluble fraction by centrifugation Whereas inclusion bodies will sediment with the cell debris soluble recombinant pr
19. the protocol 6 3 2 Preparation of cleared lysates under denaturing conditions 6 3 1 Preparation of cleared lysates under native conditions E coli e Prepare the LEW buffer Lysis Equilibration Wash Buffer and the Elution Buffer as described in 3 4 Buffer compositions e Harvest the cells from expression culture by centrifugation 15 min 4500 x g to 6000 x g 4 C e Remove supernatant and store pellet at 20 C or proceed immediately Please perform all steps on ice e Thoroughly resuspend the pellet in LEW buffer 1 25 5 mg polyhistidine tagged protein 1 ml LEW see also table 3 below by pipetting up and down or vortexing until complete resuspension is reached no cell aggregates visible anymore Add lysozyme to a final concentration of 1 mg ml and optional protease inhibitor e g 0 1 mM PMSF final concentration Stir the solution on ice for 30 min e Sonicate the suspension on ice according to the instructions provided by the manufacturer e g 10 x 15 sec burst with 20 sec rest amplitude of 20 e 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 additional 15 min e Centrifuge the crude lysate at 10000 x g for 30 min at 4 C to remove cellular debris Carefully transfer the supernatant to a clean tube without disturbing the pellet e f the supernatant is not clear centrifuge a second time or fil
20. the tag is not completely accessible e g under native conditions it will bind as long as more than two histidine residues are available for interaction In general the smaller the number of accessible histidine residues the weaker the binding MoBiTec GmbH Germany Phone 49 551 70722 0 Fax 49 551 70722 22 E Mail info mobitec com www mobitec com MoBiTec GmbH 2015 5 2 Binding capacity The maximal binding capacity of MoBiTec Ni IDA Columns is 90 mg recombinant protein as tested for histidine tagged green fluorescent protein 6xHis GFP 32 kDa The actual obtained yield is depending on the concentration of the histidine tagged fusion protein and its overall amount in the loaded sample For a maximal recovery gt 80 we recommend loading up to 75 mg polyhistidine tagged protein within 2 3 ml sample volume To obtain a maximum yield with lower recovery values we recommend loading even higher amounts of polyhistidine tagged protein up to 100 mg high concentration and a high overall amount will result in the highest possible yield 5 3 Purification of secretory proteins Producing proteins by secretion can be a great benefit in case of proper folding disulfide bond formation and for directing toxic proteins out of the cell In addition purification might be easier since the proteins can be purified directly from the corresponding compartment periplasmic space or culture medium showing a lower amount of total protein
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