ProteoSpin Inclusion Body Protein Isolation
Contents
1. approximate pH 12 5 lt 70 100 mM sodium borate approximate pH 12 5 95 100 1 M Tris approximate pH 12 5 95 Appendix 2 Proteins with Established Isoelectric Points Protein Molecular Weight kDa Isoelectric Point pl Albumin bovine serum 67 5 5 Albumin human serum 66 5 4 8 Carbonic anhydrase 30 7 3 Carboxypeptidase 34 6 0 Catalase 250 5 6 Cytochrome C 13 10 6 Fibrinogen 330 5 5 Growth hormone human 21 5 6 9 Hemoglobin horse 65 6 9 Immunoglobulin G 150 6 4 7 2 Insulin 5 7 5 3 Lysozyme hen egg white 14 3 11 0 Myoglobin horse 17 7 0 Ovalbumin 40 4 6 Pepsin 35 5 lt 1 0 Ribonuclease 14 7 8 Thyroglobulin 660 4 6 Trypsin inhibitor soybean 22 5 4 55 Urease 480 5 1 Related Products Product ProteoSpin Inclusion Body Isolation Maxi Kit 4 samples 17700 Inclusion Body Solubilization Reagent 25 mL 100 mL 18700 18701 Cell Lysis Reagent 100 mL 500 mL 18800 18801 Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2007 Norgen Biotek Corp P2. 110300 7 10
3. 3430 Schmon Parkway b lt Thorold ON Canada L2V 4Y6 gt Phone 866 667 4362 905 227 8848 Fax 905 227 1061 a amas w re any Email techsupport norgenbiotek com Inclusion Body Isolation Micro Kit Product Insert Product 10300 10600 The ProteoSpin Inclusion Body Isolation Micro Kit facilitates the screening of E coli clones that express recombinant proteins in inclusion bodies The kit includes reagents specially formulated to achieve rapid and high quality purification of inclusion body proteins using three processes 1 Lysis of bacterial cells to release inclusion bodies in solid form 2 Solubilization of purified inclusion bodies 3 Purification of the recombinant protein using spin column chromatography With optimized reagents and streamlined processes the ProteoSpin Inclusion Body Isolation Micro Kit significantly reduces time and labour for screening and identifying clonal cell lines that can be used in scale up production The ProteoSpin Inclusion Body Isolation Micro Kit employs spin column chromatography using Norgen s proprietary resin as an ion exchanger Each spin column is able to purify 2 50 ug of recombinant proteins from 1 5 mL of culture The kit is designed to purify both acidic and basic proteins Kit Components Component Product 10300 Product 10600 20 Samples 50 Samples Cee 30 mL 60 mL Wash Buffer Basio 30 mL 60 mL pH Binding B
4. The supernatant may be saved in a fresh microcentrifuge tube for comparative analysis of the soluble proteins present in this fraction 7 Using the needle and syringe technique described in step 5 again add 200 uL of Cell Lysis Reagent to the tube and carefully resuspend the pellet A few passes through the needle is sufficient to prepare a homogeneous suspension You can use the same needle and syringe from step 5 8 Prepare a 10 fold dilution of the Cell Lysis Reagent mix one part of the stock Cell Lysis Reagent to nine parts of sterile deionized water or MilliQ water Add 600 uL of this solution to the suspension prepared in step 7 and pass through the needle a few times 9 Centrifuge for 10 minutes and discard supernatant Important Use caution to avoid accidental removal of pelleted material 10 Add 800 uL of the diluted Cell Lysis Reagent to the pellet and resuspend using the needle and syringe until homogeneous 11 Centrifuge for 10 minutes and discard supernatant 12 Ensure that the pellet is relatively dry by tapping out residual liquid or by careful use of aspiration Protocol Two Solubilization of Inclusion Bodies Acidic and Basic Proteins The ProteoSpin IB Solubilization Reagent has demonstrated an exceptional ability to dissolve inclusion bodies This step is necessary before proceeding with the purification of the recombinant protein using the ProteoSpin chromatography technology Cell lysis and inclusio
5. by vortexing This step should bring the pl of your sample to 4 5 Column Activation 1 Assemble a spin column with a provided collection tube Open the cap on the column Note The collection tube will be used for the binding and wash steps Add 250 uL of Column Activation and Wash Buffer Acidic to the column and close the cap Centrifuge for 1 minute at maximum speed and discard the flowthrough Repeat steps 2 and 3 to complete the column activation step Protein Binding 1 Apply the 257 5 uL of prepared protein sample from the Sample Preparation step onto the activated column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube Note The flowthrough can be saved in a fresh tube not supplied to asses the binding efficiency of the protein Column Wash 1 Apply 250 uL of Column Activation and Wash Buffer Acidic to the column and centrifuge for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 1 and 2 Inspect the column and ensure that all the liquid has passed through into the collection tube There should be no liquid in the column If necessary spin for an additional minute to dry Protein Elution and pH Adjustment The Elution Buffer that is supplied is 50 mM sodium phosphate pH 12 5 Please refer to Appendix 1 Optional Elution Buffers for a list of alternate elution solutions that have been
6. e deionized water or Milli Q water Procedure This section describes the procedure for the ProteoSpin Inclusion Body Isolation Micro Kit and how to select the appropriate protocol for your sample Overview The Proteospin Inclusion Body Isolation Micro Kit uses a proprietary cell lysis reagent to selectively lyse the cells and release inclusion bodies in their solid form Using the IB Solubilization Reagent inclusion bodies are dissolved and their contents released Inclusion body proteins are then further purified by loading onto spin columns containing SiC Non specifically bound components in the solution can be washed from the column leaving the inclusion body protein bound to the SiC These specific proteins can then be recovered using the elution buffer Each spin column is able to recover up to 50 ug of acidic or basic protein Choosing a ProteoSpin Procedure Acidic or Basic Protocol The kit includes solutions for isolating inclusion bodies containing either acidic or basic proteins In theory the protocol for acidic proteins should apply to the majority of proteins since the resin is a cation exchanger All proteins with a pl greater than the binding pH at 4 5 should bind Basic proteins however bind strongly when they are used under these conditions making their elution quite inefficient Therefore for soluble basic proteins pl 2 8 a different condition for binding the protein to the resin has been developed For the p
7. ed protein is not used immediately degradation will occur We strongly suggest adding Neutralizer to is Degraded quickly enough lower the pH Proteases may be Use protease inhibitors during all steps of sample present preparation Bact ria contaninatisn Prepare the protein samples with 0 015 sodium f azide The Elution Buffer already contains sodium of the protein solution azide ted idl cel Inefficient cell lysis See the Problem Inefficient Cell Lysis table Appendix 1 Optional Elution Buffers Proteins bound to SiC via interactions with electrostatic charges are eluted through pH dependent mechanisms The efficiency of protein elution depends on high pH above the pl of the protein to be purified The pH of the elution buffer chosen must be at least one unit higher than the pl isoelectric point of the protein of interest Solutions not provided with the ProteoSpin Inclusion Body Isolation Micro Kit may be utilized if they are more appropriate for your needs The table below describes optional elution buffers and their observed efficiency when BSA is used as a test protein 7 i Protein Elution Buffers AER ote 50 mM ammonium hydroxide approximate pH 11 70 250 mM ammonium hydroxide approximate pH 11 70 1 M ammonium hydroxide approximate pH 11 90 1 M ethanolamine approximate pH 9 70 80 50 mM sodium phosphate approximate pH 12 5 95 500 mM sodium phosphate
8. ein too concentrated Vortex and repeatedly pipette to try and create a homogeneous solution If needed heat slightly to return the protein into solution pH of eluted protein is close to the protein s pl Check the pH to verify it is the same as the pl of the protein Add additional Neutralizer to bring the pH away from the pl of the protein 1 pH lower or higher Problem Causes Solution and Explanation Poor Protein Recovery Incorrect procedure was used Ensure that the acidic protocol was used for an acidic protein and the basic protocol for a basic protein It is known that when basic proteins are bound using the acidic protocol elution is inefficient because the basic proteins are bound tightly Incorrect pH adjustment of sample Ensure that the pH of the sample is 4 5 for acidic proteins and 7 0 for basic proteins Protein may have precipitated prior to loading onto the column If the pH of the protein sample is the same as the pl of your protein precipitation may occur In this case adjust the pH of your sample to at least one pH unit lower than the pl of your protein Eluted Protein Eluted protein solution was not neutralized Add 5 uL of Neutralizer to each 50 uL of eluted protein in order to adjust the pH to neutral Some proteins are sensitive to high pH such as the elution buffer at pH 12 5 Eluted protein solution was not neutralized If elut
9. eny Add lysozyme to concentrations recommended by the supplier Mechanical disruption of cells was inefficient Increase the number of passages through the needle and syringe Centrifugation speed was too low Check the centrifuge to ensure that it is capable of generating 14 000 x g Sufficient centrifugal force is required to move the liquid phase through the resin Inadequate spin time Spin an additional minute to ensure that the liquid is able to flow completely through the column Protein Protein solution is too Dilute the protein solution and adjust the pH Solution Does f accordingly Highly viscous materials due to high Not Flow VISCOUS protein concentration can retard the flow rate Through the f Column Cellular debris is Filter the sample in a 0 45 uM filter or spin down present in protein insoluble materials and transfer the liquid portion to solution the column Solid insoluble materials can cause f severe clogging problems Protein solution is not Dissolve the sample in a larger volume of buffer x Solid insoluble materials can cause severe clogging completely dissolved problems a Increase the degree of mechanical disruption by Spin of Cell Liberation of nucleic passing bacteria lysis reagent through the needle at Lysis is Too acids following lysis least five more times Alternatively add an Viscous appropriate amount of DNasel Eluted Protein Forms Precipitate Prot
10. n body isolation must be completed before starting the solubilization process 1 Add 50 uL of IB Solubilization Reagent to pelleted inclusion bodies 2 Dissolve the pellet by pipetting and vortexing Note If the pellet is not completely dissolved after ample pipetting and vortexing add more IB Solubilization Reagent 3 Once the pellet is dissolved add 50 uL of sterile deionized water or a volume equal to the amount of IB Solubilization Reagent used in Step 1 and mix by vortexing Now purify the recombinant protein of interest using either the acidic or basic purification procedure Protocol Three Purification of Basic Inclusion Body Proteins Proteins with isoelectric points pl greater than 7 are considered basic however proteins with a pl greater than or equal to 8 should be treated as basic proteins when using the Proteospin Inclusion Body Isolation Micro Kit If the pl of the protein being purified is not known the theoretical pl may be calculated using the web based application at http us expasy org tools pi_tool html or http www up univ mrs fr wabim d_abim compo p html Each column is capable of processing up to 50 ug of protein Sample Preparation 1 2 3 Transfer 50 uL of the dissolved protein sample to a fresh microcentrifuge tube Add 200 uL deionized or Milli Q water Prepare the protein sample by adding 7 5 uL of pH Binding Buffer Basic to the sample and mix by vortexing Column Activa
11. ne expression vector utilized The user must consult expression system instructions literature for proper use To ensure the option of scaling up clones found to contain the protein of interest it is recommended that the user preserve stocks of uninduced bacteria for each clone tested All centrifugation steps are carried out at 14 000 x g in a benchtop microcentrifuge Please check your microcentrifuge specifications to ensure proper speed All centrifugation steps are performed at room temperature Centrifugation at 4 C will not adversely affect performance Cell Lysis and Isolation of Inclusion Bodies Acidic and Basic Proteins 1 Atthe end of the protein induction period transfer 1 5 mL of the bacterial culture into a microcentrifuge tube 2 Centrifuge for one minute and discard supernatant 3 Freeze pellet at 20 C or lower using liquid N Then thaw at room temperature or at 37 C to improve lysis efficiency 4 Add 200 uL of Cell Lysis Reagent to the bacterial pellet 5 Assemble a needle with a 1 mL syringe provided Carefully disrupt the bacterial pellet by drawing it along with the Cell Lysis Reagent through the needle and ejecting the suspension back into the microcentrifuge tube Pass through the needle 15 to 20 times 6 Centrifuge the suspension for 10 minutes and carefully discard supernatant Important This supernatant may be quite viscous Do not disturb the pelleted material when discarding the supernatant
12. rocentrifuge tube Transfer the spin column from the Column Wash procedure into the microcentrifuge tube Apply 25 uL of Elution Buffer to the column and centrifuge for 1 minute to elute bound protein Add another 25 uL of Elution Buffer and centrifuge for 1 minute into the same microcentrifuge tube Note Approximately 95 of bound protein is recovered in the first two elutions If desired a third elution using 50 uL of Elution Buffer may be carried out This should be collected into a different tube to which 5 uL of Neutralizer is pre added to prevent dilution of the first two elutions Protein samples are now ready for downstream applications Protocol Three Purification of Acidic Inclusion Body Proteins Proteins with isoelectric points pl less than 7 are considered acidic however proteins with pl of less than 8 may be treated as acidic when using the Proteospin Inclusion Body Isolation Micro Kit If the pl of the protein being purified is not known the theoretical pl may be calculated using the web based application at http us expasy org tools pi_tool htm or http www up univ mrs ft wabim d_abim compo p html Each column is capable of processing up to 50 ug of protein Sample Preparation 1 2 Transfer 50 uL of the dissolved protein sample to a fresh microcentrifuge tube Add 200 uL deionized or Milli Q water Prepare the protein sample by adding 7 5 uL of pH Binding Buffer Acidic to the sample and mix
13. tested with the kit Add 5 uL of Neutralizer to a fresh 1 7 mL microcentrifuge tube Transfer the spin column from the Column Wash procedure into the microcentrifuge tube Apply 25 uL of Elution Buffer to the column and centrifuge for 1 minute to elute bound protein Add another 25 uL of Elution Buffer and centrifuge for 1 minute into the same microcentrifuge tube Note Approximately 95 of bound protein is recovered in the first two elutions If desired a third elution using 50 uL of Elution Buffer may be carried out This should be collected into a different tube to which 5 uL of Neutralizer is pre added to prevent dilution of the first two elutions Protein samples are now ready for downstream applications Troubleshooting Guide Problem Causes Solution and Explanation Improper induction of Consult the manufacturer s expression system No inclusion gene expression literature body pelapis Gene product does not observed produce inclusion bodies Reassess the expression cassette Inefficient Cell Lysis Kit solutions were improperly stored Keep the lysis and solubilization reagent at 4 C at all times when not in use The two binding buffers are kept at room temperature Freeze thaw step was not performed The freeze thaw step is known to increase lysis efficiency Repeat the protocol using the recommended freeze thaw conditions Lysozyme may be required to increase lysis effici
14. tion 1 Assemble a spin column with a provided collection tube Open the cap on the column Note The collection tube will be used for the binding and wash steps Add 250 uL of Column Activation and Wash Buffer Basic to the column and close the cap Centrifuge for 1 minute at maximum speed and discard the flowthrough Repeat steps 2 and 3 to complete the column activation step Protein Binding 1 Apply the 257 5 uL of prepared protein sample from the Sample Preparation step onto the activated column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube Note The flowthrough can be saved in a fresh tube not supplied to asses the binding efficiency of the protein Column Wash 1 Apply 250 uL of Column Activation and Wash Buffer Basic to the column and centrifuge for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 1 and 2 Inspect the column and ensure that all the liquid has passed through into the collection tube There should be no liquid in the column If necessary spin for an additional minute to dry Protein Elution and pH Adjustment The Elution Buffer that is supplied is 50 mM sodium phosphate pH 12 5 Please refer to Appendix 1 Optional Elution Buffers for a list of alternate elution solutions that have been tested with the kit 1 2 3 Add 5 uL of Neutralizer to a fresh 1 7 mL mic
15. uffer Acidic 1 mL 1 mL pH Binding Buffer Basic 1 mL 1 mL Elution Buffer 4mL 8 mL Neutralizer 1 mL 1 mL Cell Lysis Reagent 15 mL 30 mL IB Solubilization Reagent 2mL 4mL Syringes 1cc slip tip 20 50 Needles Bev 20G x 1 inch 20 50 Micro Spin Columns 20 50 Collection Tubes 20 50 Elution tubes 1 7 mL 20 50 Product Insert 1 1 Storage Conditions and Product Stability The Cell Lysis and IB Solubilization Reagents should be stored at 4 C upon receipt of this kit For other unopened solution containers the reagents should remain stable for 1 year when stored at room temperature Once opened the solutions should be stored at 4 C when not in use except for the pH Binding Buffers Acidic and Basic Some precipitation will occur with 4 C storage This precipitation should be dissolved with slight heating to room temperature before using Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Customer Supplied Reagents and Equipment You must have the following in order to use the ProteoSpin Inclusion Body Isolation Micro Kit e Benchtop microcentrifuge e Micropipettors e Steril
16. urposes of the Proteospin Inclusion Body Isolation Micro Kit a protein with a pl less than 8 will be treated as acidic and will use the acidic protocol For a protein with a pl greater than or equal to 8 use the basic protocol 1 Follow the Procedure for Basic Proteins Net I Charge Of L l Protein 2 Follow the Procedure for i Acidic Proteins j ai L A Isoelectric Point Protocols To rapidly screen bacterial clones for expression of recombinant proteins in inclusion bodies the ProteoSpin Inclusion Body Isolation Micro Kit is designed for testing small bacterial cultures growing in test tubes Test tube cultures with a 2 mL culture medium are normally initiated with single colonies picked from culture plates Protocol One Isolating Inclusion Bodies For both Acidic and Basic Proteins The procedure for lysing bacterial cells to release their inclusion bodies is identical for all recombinant proteins to be screened The procedure for purifying proteins from solubilized inclusion bodies using ProteoSpin columns however depends on the isoelectric point of the recombinant protein that is expressed in the inclusion bodies The user must decide whether to use the acidic or basic procedures depending on the pl of the recombinant protein in question The efficiency of inclusion body extraction may vary from strain to strain Growth and induction conditions are dependent upon host strain and ge
Download Pdf Manuals
Related Search