Home

Protocol

1. 3 Purification of GST tagged Proteins User Manual Protino GST 4B Columns 1 ml Protino GST 4B Columns 5 ml January 2010 Rev 01 MACHEREY NAGEL MN Purification of GST tagged Proteins Table of contents 1 Components 4 1 1 Kit contents and storage 4 1 2 Reagents consumables and equipment to be supplied by the user 4 2 Introduction 6 2 1 Specifications 6 2 2 Culture size 7 2 3 Preparation of buffers 9 3 Safety instructions risk and safety phrases 11 4 Protocols 12 4 1 Preparation of cleared E coli lysates 12 4 2 Purification of GST tagged proteins using Protino GST 4B Columns 14 5 Regeneration and storage 16 6 Appendix 17 6 1 Troubleshooting 17 6 2 Ordering information 19 6 3 Product use restriction warranty 20 MACHEREY NAGEL 01 2010 Rev 01 3 o _D Purification of GST tagged Proteins 1 Components 1 1 Kit contents and storage Protino GST 4B Columns 1 ml 5 ml Cat No 745510 5 745515 1 745515 5 Protino GST 4B Columns 5x1 ml 1x5 ml 5x5ml User Manual 1 1 1 Shipping and storage of Protino GST 4B Columns The product is shipped at ambient temperature Upon receipt Protino GST 4B Columns should be stored at 4 C and are stable up to 1 year Do not freeze 1 2 Reagents consumables and equipment to be supplied by the user Reagents e PBS 10 mM Na HPO 1 8 mM KH PO 2 7 mM KCI 140 mM NaCl pH 7 3 see section 2 3 e Elution Buffer 50 mM Tr
2. of 100 mM Tris HCI 0 5 M NaCl pH 8 5 followed by approximately 10 column volumes of 100 mM sodium acetate 0 5 M NaCl pH 4 5 Repeat the above wash cycles twice Wash with 5 column volumes of PBS Rigorous cleaning To remove precipitated or denatured proteins wash with 2 column volumes of 6 M guanidine hydrochloride immediately followed by 5 10 column volumes of PBS To remove hydrophobically bound contaminants wash with 4 column volumes of 70 ethanol or 1 Triton X 100 followed by 5 10 column volumes of PBS If you will not be using the matrix immediately wash with additional 5 column volumes of 20 ethanol and store at 4 C 16 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Problems with vector construction e Ensure that protein and tag are in frame Low protein expression e Optimize bacterial expression conditions a ie protein Fusion protein forms insoluble aggregates inclusion bodies y e Lower the growth temperature from 37 C to 30 15 C Extraction may be insufficient e Check extraction conditions lysozyme sonication e Use up to 2 of a non ionic detergent to improve cell disruption and or solubilization of the fusion protein Sonication may have been to severe e Choose milder sonication conditions Over sonication can alter the conformation of the GST moiety and prevents the fusion protein from
3. proteins is high ranging from 10 to 50 mg liter of E coli culture e Asa starting point we recommend to use the cell lysate from a 200 1000 ml E coli culture per 1 ml bed volume 2 Slow flow rates are recommended for the loading step to allow maximal binding of the GST tagged protein MACHEREY NAGEL 01 2010 Rev 01 7 Purification of GST tagged Proteins Protino GST 4B Columns 1 ml bed volume Load 10 mg protein JN Expression level Expression level 10 mg l 50 mg l Use 1000 ml culture Use 200 ml culture 4 g cell pellet 0 8 g cell pellet Resuspend in Resuspend in up to 20 ml PBS up to 4 ml PBS 24 ml protein lysate 5 ml protein lysate Figure 1 Required culture volumes per 1 ml bed volume 1 On average 250 ml of culture will produce approximately 1 g of pelleted wet cells 2 1 g cells may be lysed in 2 5 ml PBS see section 4 1 8 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins 2 3 Preparation of buffers PBS 1 liter 10 mM NaH PO 1 780 g Na HPO 2 H O M 156 01 g mol 1 8 mM KH PO 0 245 g KH PO M 136 09 g mol 2 7 mM KCI 0 201 g KCI M 74 55 g mol 140 mM NaCl 8 182 g NaCl M 58 44 g mol Adjust pH to 7 3 Elution Buffer 1 liter 50 mM Tris base 6 06 g Tris base M 121 14 g mol 10 mM glutathione 3 07 g glutathione M 307 3 g mol Adjust pH to 8 0 Prepare fresh daily and store at 4 C Minimum buff
4. pump 1 16 ID 1 x 1 16 ID tubing to 10 32 male Protino Inlet PP tubing Adaptor Set inlet Cat No 745263 Syringe Luer 1 x Luer female to 10 32 male Protino Inlet Luer inlet Adaptor Cat No 745262 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins 2 Introduction Protino GST 4B Columns are convenient ready to use FPLC columns prepacked with Protino Glutathione Agarose 4B for rapid purification of GST fusion proteins The columns can be used with an automated chromatography system a peristaltic pump or with a syringe for manual processing Protino GST 4B Columns can be attached directly to liquid chromatography systems such as AKTAdesign systems via standard 10 32 fittings The columns can also be operated with other chromatography systems with a syringe or peristaltic pump by using common adapters provided separately by MN for details see section 1 2 or contact Technical Service Bioanalysis The snap off end of the columns can be reused as stop plug for sealing the outlet of the columns for storage 2 1 Specifications Table 2 Specifications Protino GST 4B Columns Column bed volume 1ml 5 ml System compatibility Automated liquid chromatography systems MPLC FPLC AKTAdesign etc Peristaltic pump Syringe Column dimensions 0 7 cm inner diameter 1 6 cm inner diameter x 2 5 cm height x 2 5 cm height Column body m
5. 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 20 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins 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 Last update
6. aterial Polypropylene Column ports Inlet 10 32 1 16 female Outlet 10 32 1 16 male Matrix 4 beaded agarose Ligand Glutathione linked via sulfur atom Spacer arm 12 atoms Bead size 90 um Binding capacity 10 mg 50 mg recombinant GST recombinant GST 1 Binding capacity will vary for each GST tagged protein 6 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins Table 2 Specifications Protino GST 4B Columns Maximum back pressure 3 bar 0 3 MPa Recommended flow rates Equilibration 1 0 ml min 2 5 ml min Sample loading 0 2 1 0 ml min 0 5 2 0 ml min washing and elution 1 0 ml min 5 0 ml min Chemical stability Protino GST 4B Columns withstand incubation in 0 1 M acetate pH 4 0 1 M NaOH 70 ethanol or 6 M guanidine hydrochloride for 2 hours at room temperature without significant loss of protein yield Storage temperature 4 8 C Storage solution 20 ethanol 2 2 Culture size The yield of GST tagged proteins depends on various parameters such as nature of the fusion protein expression host culture conditions etc However some recommendations on protein load and culture size can be given see Figure 1 Culture volume requirements are based on the following assumptions e Protino GST 4B Columns have a binding capacity of 10 mg of recombinant GST per 1 ml bed volume e Typically the expression level of GST tagged
7. binding to Protino GST 4B Columns Reducing agent missing e Adding DTT to the lysis buffer final concentration 5 mM prior Fusion to cell lysis can significantly increase binding of some fusion protein does proteins not bind efficiently Flow rate too high e Decrease flow rate for the loading step to allow maximal binding of the GST tagged protein Concentration of fusion protein is too dilute e Concentrate the sample Yield depends on the concentration of the fusion protein in the lysate If the sample is too dilute fusion proteins may not bind efficiently MACHEREY NAGEL 01 2010 Rev 01 17 Purification of GST tagged Proteins Problem Possible cause and suggestions Fusion Protino GST 4B Columns have been used several times protein does e Clean matrix according to section 5 or use fresh matrix not bind Immobilized glutathione can be degraded by y glutamyl efficiently transpeptidase activity in E coli cell lysates Therefore matrices continued with immobilized glutathione have a finite lifetime Low elution volume e Increase the volume of Elution Buffer Depending on the nature of the fusion protein and the amount of protein loaded additional elution steps or buffer volume is required Flow rate too high Fusion e Decrease flow rate during elution protein elutes inefficiently Incorrect buffer composition e Check composition and pH of the Elution Buffer In some cases up to 50mM reduced glutathione ma
8. d 12 2006 Rev 02 Trademarks AKTAdesig and FPLC are trademarks of GE Healthcare companies BioLogic and Profinia are trademarks of BIO RAD Laboratories Inc 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 01 2010 Rev 01 21
9. e to a final concentration of 1 mg ml e Stir the solution on ice for 30 min e 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 e 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 ice for 15 min 12 MACHEREY NAGEL 01 2010 Rev 01 Protino GST 4B Columns 4 Clarify lysate e Centrifuge the crude lysate at 10 000 x g for 30 min at 4 C to remove cellular debris e 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 cellulose acetate e Store supernatant on ice Proceed to section 4 2 MACHEREY NAGEL 01 2010 Rev 01 13 Protino GST 4B Columns 4 2 Purification of GST tagged proteins using Protino GST 4B Columns Protino GST 4B Columns can be operated with liquid chromatography systems such as AKTAdesign systems via standard 10 32 fittings without additional connectors Prepare buffers according to section 2 3 For best results filter buffers through a 0 45 um filter before use Clear samples by centrifugation and or pass them through a 0 45 um filter Binding kinetics between GST and immobilized glutathione is relatively slow Therefore
10. er volumes required for one column run can be calculated according to the following table Note that additional volumes of PBS and Elution Buffer may be prepared to flush lines and pumps depending on the chromatographic system As a starting point we recommend to prepare approximately 150 ml of PBS and 100 ml of Elution Buffer for the 1 ml column and 300 ml of PBS and 150 ml of Elution Buffer for the 5 ml column Use high purity chemicals and water for preparing the buffers For best results filter buffers through a 0 45 um filter before use Protino GST 4B Columns imi 5 ml PBS 5 ml per 1 g of cell pellet for cell extract preparation 20 ml 100 ml 10 ml per 1 ml bed volume for equilibration 10 ml 50 ml 10 ml per 1 ml bed volume for washing 10 ml 50 ml 50 ml of PBS for flushing lines and pump 50 ml 50 ml Total volume of PBS 90 ml 250 ml Recommended volume of PBS 150 ml 300 ml MACHEREY NAGEL 01 2010 Rev 01 9 Purification of GST tagged Proteins Protino GST 4B Columns 1ml 5 ml Elution Buffer 10 ml per 1 ml bed volume for equilibration 10 ml 50 ml 50 ml of Elution Buffer for flushing lines and pump 50 ml 50 ml Total volume of Elution Buffer 60 ml 100 ml Recommended volume of Elution Buffer 100 ml 150 ml 10 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins 3 Safety instructions risk and safety phrases The following components of the Protino GST 4B Columns kits con
11. is HCI 10 mM glutathione pH 8 see section 2 3 e Lysozyme required for cell extract preparation see section 4 1 Consumables e Appropriate centrifuge tubes collecting tubes Equipment e Appropriate centrifuge e Liquid chromatography system MPLC FPLC AKTAdesign etc peristaltic pump or syringe e Ifnecessary appropriate adaptors for connecting the Protino GST 4B Columns to your system of choice Protino GST 4B Columns are equipped with 10 32 1 16 inlet and outlet ports With these ports the columns can easily be connected to standard MPLC FPLC systems e g AKTAdesign Five 4 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins adaptor sets are available for connecting the columns to other systems or for using them with a syringe see Table 1 Table 1 Adaptor sets System Connec Adaptor needed Adaptor Set tion via Standard FPLC 10 32 none none system e g AKTAdesign FPLC system M6 1 x M6 female to 10 32 male Protino M6 first generation 1 x 10 32 female to M6 male Adaptor Set Pharmacia Cat No 745260 MPLC system 1 4 28 1 x 1 4 28 female to 10 32 male Protino 1 4 28 e g BioLogic 1x 10 32 female to 1 4 28 Adaptor Set BIO RAD female Cat No 745261 MPLC system Luer 1 x Luer female to 10 32 male Protino Luer e g BioLogic 1 x 10 32 female to Luer male Adaptor Set BIO RAD Cat No 745264 Peristaltic
12. omer 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 products 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
13. t flow through and analyze for example by SDS PAGE to verify that the GST tagged protein has bound If the fusion protein is found in early fractions of the flow through the flow rate should be decreased If the fusion protein is absent in early fractions and does appear in late fractions of the flowthrough the column capacity has been exceeded In this case protein load should be reduced or bed volume should be increased Washing e Wash the column with 10 column volumes of PBS or until the baseline at 280 nm is stable 10 ml 50 ml Use a flow rate up to 1 ml min 5 ml min Elution e lute the GST tagged protein with 10 column volumes of Elution Buffer and collect fractions 10 ml 50 ml Use a flow rate up to 1 ml min 5 ml min e Use a Bradford protein assay SDS PAGE or measure the absorbance at 280 nm to identify the fraction s which contain s the majority of the eluted GST tagged protein and analyze by SDS PAGE MACHEREY NAGEL 01 2010 Rev 01 15 Purification of GST tagged Proteins 5 Regeneration and storage Reuse of Protino GST 4B Columns should only be performed with identical GST tagged proteins to avoid possible cross contamination The lifetime of the matrix depends on the nature of the sample If a single GST tagged protein is to be purified several times simply wash with 10 column volumes of PBS prior to the next column run Basic cleaning Wash column with approximately 10 column volumes
14. tain hazardous contents Component Hazard Hazard Risk Safety contents symbol phrases phrases Protino GST 4B Columns Ethanol 20 i Flammable R10 1ml and 5 ml Risk phrases R10 Flammable Hazard labeling not necessary if quantity per bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet MACHEREY NAGEL 01 2010 Rev 01 11 Protino GST 4B Columns 4 Protocols 4 1 Preparation of cleared E coli lysates Refer to sections 2 2 for detailed information on culture volume requirements Prepare PBS as described at section 2 3 e 1 Cultivate and harvest cells e Asastarting point we recommend to prepare 200 1000 ml E coli expression culture for the purification of 10 mg of GST tagged protein per 1 ml bed volume using Protino GST 4B Columns see section 2 2 e 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 e Cell pellets may be stored at 20 C or 80 C until needed 2 Resuspend bacteria cells e Thaw the cell pellet from an E coli expression culture on ice if frozen e Resuspend 1 g of pelleted wet cells in 2 5 ml PBS Pipette up and down or stir until complete resuspension without visible cell aggregates Perform this step on ice 3 Lyse cells e Add lysozym
15. ts in the presence of ATP and Mg 6 2 Ordering information Product Cat No Pack of Protino GST 4B Columns 1 ml 745510 5 5 columns Protino GST 4B Columns 5 ml 745515 1 1 column 745515 5 5 columns Protino Glutathione Agarose 4B 745500 10 10 ml settled agarose beads 745500 100 100 ml settled agarose beads Protino M6 Adaptor Set 745260 1 set Protino 1 4 28 Adaptor Set 745261 1 set Protino Luer Adaptor Set 745264 1 set Protino Inlet PP Adaptor Set 745263 1 set Protino Inlet Luer Adaptor 745262 1 Visit www mn net com for more detailed product information MACHEREY NAGEL 01 2010 Rev 01 19 Purification of GST tagged Proteins 6 3 Product use restriction warranty Protino GST 4B Columns were developed designed distributed 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 the Protino GST 4B Columns 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 cust
16. use low flow rates for the loading step to allow maximal binding of the GST tagged protein Prepare Elution Buffer as described at section 2 3 Elution Buffer has to be prepared fresh daily and stored at 4 C e Protino GST 4B Columns 1 Connect column to the chromatography system e Purge the pump with PBS Assure that all air is displaced e Remove the snap off end at the column outlet and save it for further use e Remove the upper plug from the column e Start the pump at a flow rate of approximately 0 3 ml min e Fill the inlet port of the column with several drops of PBS to remove air to form a positive meniscus e Insert the fitting drop to drop into the column port to avoid introducing air bubbles Note The snap off end can be reused as a stop plug for sealing the column outlet for storage 2 Column equilibration e Equilibrate the column with 5 10 column volumes of PBS until the baseline at 280 nm is stable 5 10 ml 50 100 ml Use a flow rate up to 1 ml min 2 5 ml min 14 MACHEREY NAGEL 01 2010 Rev 01 Protino GST 4B Columns Protino GST 4B Columns 1 ml Binding e Load the centrifuged or filtered sample onto the column e Use a flow rate up to 0 2 1 0 ml min 0 5 2 ml min Note Binding kinetics between GST and immobilized glutathione is relatively slow Therefore use low flow rates for the loading step to allow maximal binding of the GST tagged protein e Collec
17. y be used to improve elution Elution Buffer not prepared immediately before use e Prepare Elution Buffer immediately before use Poor protein purity Insufficient washing e Increase the number of washes with PBS Degradation of GST fusion protein e Adda protease inhibitor to the lysis solution Multiple bands may be the result of partial degradation by host proteases during the purification procedure e Keep all samples and buffers on ice to reduce the activity of proteases e Use a protease deficient host Multiple bands may be the result of partial degradation by host proteases during cell growth Sonication may have been too severe e Choose milder sonication conditions Over sonication can lead to the co purification of host proteins with the GST tagged protein 18 MACHEREY NAGEL 01 2010 Rev 01 Purification of GST tagged Proteins Problem Possible cause and suggestions Co purification of chaperonins e Several chaperonins that are involved in protein folding may co purify with GST fusion proteins for example Dnak Poor protein purity continued 70 kDa DnaJ 37 kDa GrpE 40 kDa GroEL 57 kDa GrpE 40 kDa GroEL 57 kDa GroES 10 kDa Several additional purification steps have been described For example co purification of DnaK can be avoided by treating the cell lysate with 5 mM MgCl and 5 mM ATP prior to purification DnaK can be dissociated from other componen

Protocol

Contents

Download Pdf Manuals

Related Search

Related Contents