17150 - Protocol - Norgen Biotek Corp.
Contents
1. Follow the Procedure for 1 it I f 1 I k I I I I i i Acidic Proteins I qm i Isoelectric Point Figure 1 Choosing a procedure based on the isoelectric point pl Protocol 1 Concentration of Protein Samples and Detergent Clean up Acidic Protocol Proteins with an isoelectric point pl of less than 7 are by definition acidic proteins However for the purposes of using this kit the Protocol for Acidic Proteins applies to any protein whose pl is less than 8 0 Notes Before Use Ensure that all particulates in your sample have been removed by either filtration or centrifugation prior to starting the procedure The column reservoir has a capacity of 20 mL hence multiple centrifugations will be required for larger volumes Ensure that during each centrifugation step the cap is screwed loosely onto the column 1 Sample Preparation This step ensures that the protein solution is at the proper pH for column binding a b c e Obtain protein sample If particulates are present clarify the sample through either filtration or centrifugation Determine the pH and volume of the protein sample Adjust the pH of the protein sample to 3 5 4 for total protein concentration or to your desired pH according to the pl of your protein of interest using the Binding Buffer A and mix content well Verify that the pH is between 3 5 4 and add more Binding Buffer A if necessary Binding Buffer N can be use2. c Apply 4 mL of Elution Buffer C to the column and centrifuge for 2 minutes to elute the bound protein Note Approximately 90 of bound protein is recovered in the first two elutions If desired a second elution using Elution Buffer C may be carried out This should be collected into a different tube to which Protein Neutralizer is pre added to prevent dilution of the first elution Protocol 2 Concentration of Protein Samples Not Containing Detergent Acidic Protocol Proteins with an isoelectric point pl of less than 7 are by definition acidic proteins However for the purposes of using this kit the Protocol for Acidic Proteins applies to any protein whose pl is less than 8 0 Notes Before Use e Ensure that all particulates in your sample have been removed by either filtration or centrifugation prior to starting the procedure e The column reservoir has a capacity of 20 mL hence multiple centrifugations will be required for larger volumes e Ensure that during each centrifugation step the cap is screwed loosely onto the column 1 Sample Preparation This step ensures that the protein solution is at the proper pH for column binding a Obtain protein sample If particulates are present clarify the sample through either filtration or centrifugation b Determine the pH and volume of the protein sample c Adjust the pH of the protein sample to 3 5 4 for total protein concentration or to your desired pH according to the pl of
3. 100 mM buffered solution 4 150 uL 5 80 uL 6 80 uL 7 O uL 8 60 uL 9 60 uL 10 60 uL 11 80 uL 12 80 uL 2 Column Activation a b c Obtain a spin column with its 50 mL conical collection tube Remove lid Apply 5 mL of Wash Solution N to the column Screw the cap back on LOOSELY Centrifuge at 1 000 x g for two minutes Note Start timing only after centrifuge has reached desired speed Repeat steps 2b and 2c to complete the column activation step Discard the flowthrough 3 Protein Binding a Apply the protein sample from the Sample Preparation Step onto the column and centrifuge for two minutes The column can accommodate a maximum of 20 mL per spin Discard the flowthrough Reassemble the spin column with its collection tube Note If desired the flowthrough can be saved in a fresh tube for assessing your protein s binding efficiency Depending on your sample volume repeat steps 3a and 3b until the entire protein sample has been applied to the column Discard any remaining flowthrough and reassemble the spin column with its collection tube 4 Column Wash amii A a Apply 10 mL of Wash Solution N to the column and centrifuge for two minutes Discard the flowthrough and reassemble the spin column with its collection tube Add 10 mL of Wash Solution N to the column and centrifuge for two minutes Inspect the column and ensure that the liquid has passed through into the collection tube There s
4. 2c to complete the column activation step Discard the flowthrough 3 Protein Binding a Apply the protein sample from the Sample Preparation Step onto the column and centrifuge for two minutes The column can accommodate a maximum of 20 mL per spin Discard the flowthrough Reassemble the spin column with its collection tube Note If desired the flowthrough can be saved in a fresh tube for assessing your protein s binding efficiency Depending on your sample volume repeat steps 3a and 3b until the entire protein sample has been applied to the column Discard any remaining flowthrough and reassemble the spin column with its collection tube 4 Column Wash a a0 Apply 10 mL of Wash Solution NIP after the addition of Isopropanol to the column and centrifuge for two minutes Discard the flowthrough and reassemble the spin column with its collection tube Add 10 mL of Wash Solution N to the column and centrifuge for two minutes Inspect the column and ensure that the liquid has passed through into the collection tube There should be no liquid in the column If necessary spin an additional two minutes to dry 5 Protein Elution The Elution Buffer C that is supplied is 10 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 It is recommended that upon elution the protein solution is neutralized immediately esp
5. downstream applications such as SDS PAGE isoelectric focusing X ray crystallography NMR spectroscopy mass spectroscopy and other applications The ProteoSpin Total Protein Detergent Concentration and Clean Up Maxi Kit can remove greater than 95 of detergents from total protein samples while maintaining high protein recovery The kit is able to remove all types of detergents including ionic non ionic and zwitterionic detergents It is designed to remove detergents from protein solutions either in their free form or bound form as when complexed with the protein The ProteoSpin Total Protein Concentration and Detergent Clean Up Maxi Kit contains all the solutions and columns for the processing of 25 total protein samples Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix Each spin column is able to process small detergent containing samples from 0 25 8 mg of protein Detergents including SDS Triton X 100 CHAPS NP 40 and Tween 20 can be removed using the kit with protein recoveries of 80 95 for most proteins Preparation time for 12 samples is only 20 minutes The kit has a shelf life of at least 1 year when stored as suggested Kit Components Component Sasa Binding Buffer A 20 mL Binding Buffer N 20 mL Wash Solution C 60 mL Wash Solution CIP 60 mL Wash Solution N 60 mL Wash Solution NIP 60 mL Elution Buffer C 2x30
6. warm the solutions and mix well until the solutions become clear again e Prepare a working concentration of Wash Solution CIP by adding 60 mL of isopropanol to be provided by the user to the supplied bottle containing Wash Solution CIP This will give a final volume of 120 mL The label on the bottle has a box that can be checked to indicate that isopropanol has been added e Prepare a working concentration of Wash Solution NIP by adding 60 mL of isopropanol to be provided by the user to the supplied bottle containing Wash Solution NIP This will give a final volume of 120 mL The label on the bottle has a box that can be checked to indicate that isopropanol has been added Procedure The ProteoSpin Total Protein Concentration and Detergent Clean Up Maxi Kit comes with solutions for processing both acidic and basic proteins Two procedures one for acidic proteins and another for basic proteins are described Proteins with isoelectric points pl of less than 7 are by definition acidic proteins However for purposes of using the kit the protocol for acidic proteins applies to any protein whose pl is less than 8 0 Proteins with pl higher than 8 0 are purified using the protocol for basic proteins If the pl of the protein being purified is not known the theoretical pl may be calculated using the web based applications at http us expasy org tools pi_tool html Follow the Procedure for Basic Proteins Of L L Protein 2 4 6
7. your protein of interest using the Binding Buffer A and mix content well d Verify that the pH is between 3 5 4 and add more Binding Buffer A if necessary Binding Buffer N can be used to re adjust your pH Note 1 If the protein solution is already at the desired pH or lower Binding Buffer A does not need to be added Note 2 In some concentrated protein samples precipitation may occur with the addition of the Binding Buffer A This precipitate includes proteins and thus should not be discarded The precipitate should be resuspended as much as possible and loaded onto the column with the rest of the sample 2 Column Activation a Obtain a spin column with its 50 mL conical collection tube Remove lid b Apply 5 mL of Wash Solution C to the column Screw the cap back on LOOSELY c Centrifuge at 1 000 x g for two minutes Note Start timing only after centrifuge has reached desired speed d Repeat steps 2b and 2c to complete the column activation step Discard the flowthrough 3 Protein Binding a Apply the protein sample from the Sample Preparation Step onto the column and centrifuge for two minutes The column can accommodate a maximum of 20 mL per spin b Discard the flowthrough Reassemble the spin column with its collection tube Note If desired the flowthrough can be saved in a fresh tube for assessing your protein s binding efficiency c Depending on your sample volume repeat steps 3a and 3b until the entire
8. aining flowthrough and reassemble the spin column with its collection tube 4 Column Wash a Apply 10 mL of Wash Solution CIP after the addition of Isopropanol to the column and centrifuge for two minutes Discard the flowthrough and reassemble the spin column with its collection tube Add 10 mL of Wash Solution C to the column and centrifuge for two minutes Inspect the column and ensure that the liquid has passed through into the collection tube There should be no liquid in the column If necessary spin an additional two minutes to dry 205 5 Protein Elution The Elution Buffer C that is supplied is 10 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 It is recommended that upon elution the protein solution is neutralized immediately especially for proteins that are known to be sensitive to high pH It is therefore recommended that the Protein Neutralizer that is provided is pre added to the elution tube However this is not needed for the first elution For the second elution 0 35 mL of Protein Neutralizer is needed Note Please verify the pH of your first eluted protein sample and adjust with the Neutralizer if required a Add 350 uL Protein Neutralizer if desired not required for the 1st elution to a fresh 50 mL elution tube b Transfer the spin column with bound protein into the 50 mL elution tube from step 6a
9. d to re adjust your pH Note 1 If the protein solution is already at the desired pH or lower Binding Buffer A does not need to be added Note 2 In some concentrated protein samples precipitation may occur with the addition of the Binding Buffer A This precipitate includes proteins and thus should not be discarded The precipitate should be resuspended as much as possible and loaded onto the column with the rest of the sample Add one volume of isopropanol to the pH adjusted solution and mix well 2 Column Activation a b C Obtain a spin column with its 50 mL conical collection tube Remove lid Apply 5 mL of Wash Solution CIP after the addition of Isopropanol to the column Screw the cap back on LOOSELY Centrifuge at 1 000 x g for two minutes Note Start timing only after centrifuge has reached desired speed d Repeat steps 2b and 2c to complete the column activation step Discard the flowthrough 3 Protein Binding a Apply the protein sample from the Sample Preparation Step onto the column and centrifuge for two minutes The column can accommodate a maximum of 20 mL per spin b Discard the flowthrough Reassemble the spin column with its collection tube Note If desired the flowthrough can be saved in a fresh tube for assessing your protein s binding efficiency c Depending on your sample volume repeat steps 3a and 3b until the entire protein sample has been applied to the column d Discard any rem
10. djust the pH of the protein sample to 7 0 using the Binding Buffer N The amount of Binding Buffer N required will depend on the starting protein solution If the starting protein solution is in water then add one part of the Binding Buffer N to 50 parts of the protein solution However if the starting protein solution already contains a buffer a greater volume of Binding Buffer N may be needed depending on the sample s buffer type and strength as well as the type of protein Table 1 below serves only as a guideline for the amount of Binding Buffer N to add for every milliliter of a protein solution in a 100 mM buffer to obtain pH 7 0 Please check the pH after mixing and add more Binding Buffer N if necessary to obtain the desired pH d Table 1 pH Adjustment for Basic Proteins Volume of Binding Buffer N per mL of Starting pH of Solution protein solution based on 100 mM buffered solution 4 150 uL 5 80 uL 6 80 uL 7 O ul 8 60 uL 9 60 uL 10 60 uL 11 80 uL 12 80 uL Add one volume of isopropanol to the pH adjusted solution and mix well 2 Column Activation a b C Obtain a spin column with its 50 mL conical collection tube Remove lid Apply 5 mL of Wash Solution NIP after the addition of Isopropanol to the column Screw the cap back on LOOSELY Centrifuge at 1 000 x g for two minutes Note Start timing only after centrifuge has reached desired speed Repeat steps 2b and
11. ecially for proteins that are known to be sensitive to high pH It is therefore recommended that the Protein Neutralizer that is provided is pre added to the elution tube However this is not needed for the first elution For the second elution 0 35 mL of Protein Neutralizer is needed Note Please verify the pH of your first eluted protein sample and adjust with the Neutralizer if required a Add 350 uL Protein Neutralizer if desired not required for the 1st elution to a fresh 50 mL elution tube b Transfer the spin column with bound protein into the 50 mL elution tube from step 6a c Apply 4 mL of Elution Buffer C to the column and centrifuge for 2 minutes to elute the bound protein Note Approximately 90 of bound protein is recovered in the first two elutions If desired a second elution using Elution Buffer C may be carried out This should be collected into a different tube to which Protein Neutralizer is pre added to prevent dilution of the first elution Protocol 4 Concentration of Protein Samples Not Containing Detergent Basic Protocol Proteins with an isoelectric point pl of less than 7 are by definition acidic proteins However for the purposes of using this kit the Protocol for Acidic Proteins applies to any protein whose pl is less than 8 0 Proteins with a pl higher than 8 0 are purified using the Protocol for Basic Proteins Notes Before Use e Ensure that all particulates in your sample have been removed by
12. either filtration or centrifugation prior to starting the procedure e The column reservoir has a capacity of 20 mL hence multiple centrifugations will be required for larger volumes e Ensure that during each centrifugation step the cap is screwed loosely onto the column 1 Sample Preparation This step ensures that the protein solution is at the proper pH for column binding a Obtain protein sample If particulates are present clarify the sample through either filtration or centrifugation b Determine the pH and volume of the protein sample c Adjust the pH of the protein sample to 7 0 using the Binding Buffer N The amount of Binding Buffer N required will depend on the starting protein solution If the starting protein solution is in water then add one part of the Binding Buffer N to 50 parts of the protein solution However if the starting protein solution already contains a buffer a greater volume of Binding Buffer N may be needed depending on the sample s buffer type and strength as well as the type of protein Table 2 below serves only as a guideline for the amount of Binding Buffer N to add for every milliliter of a protein solution in a 100 mM buffer to obtain pH 7 0 Please check the pH after mixing and add more Binding Buffer N if necessary to obtain the desired pH Table 2 pH Adjustment for Basic Proteins Volume of Binding Buffer N per mL of Starting pH of Solution protein solution based on
13. g gt lt NORGEN BIOTEK wie CORPORATION 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com ProteoSpin Total Protein Concentration and Detergent Clean Up Maxi Kit Product Insert Product 17150 The ProteoSpin Total Protein Concentration and Detergent Clean Up Maxi Kit provides a fast and simple procedure for the removal of SDS Triton X 100 and other detergents from total protein samples including lysates Detergents are extensively used to prepare protein samples however these detergents must often be removed prior to downstream analysis because of their undesirable effects These include extraneous peaks in mass spectrometry artifacts with chromatography and electrophoresis interference with microinjection into cells and interference with protein immunization The ProteoSpin Total Protein Concentration and Detergent Clean Up Maxi Kit also provides a fast and simple procedure for concentrating small volumes of total protein solutions for buffer exchange and for removing different types of salts from protein samples The kit is highly efficient in removing many different salts commonly used in the laboratory including but not limited to MgCl NaCl KCI CaCl LiCl and CsCl The simultaneous removal of salts while concentrating a dilute protein solution makes the kit a convenient method for preparing proteins before running many
14. he 50 mL elution tube from step 6a c Apply 4 mL of Elution Buffer C to the column and centrifuge for 2 minutes to elute the bound protein Note Approximately 90 of bound protein is recovered in the first two elutions If desired a second elution using Elution Buffer C may be carried out This should be collected into a different tube to which Protein Neutralizer is pre added to prevent dilution of the first elution Protocol 3 Concentration of Protein Samples and Detergent Clean up Basic Protocol Proteins with an isoelectric point pl of less than 7 are by definition acidic proteins However for the purposes of using this kit the Protocol for Acidic Proteins applies to any protein whose pl is less than 8 0 Proteins with a pl higher than 8 0 are purified using the Protocol for Basic Proteins Notes Before Use Ensure that all particulates in your sample have been removed by either filtration or centrifugation prior to starting the procedure The column reservoir has a capacity of 20 mL hence multiple centrifugations will be required for larger volumes Ensure that during each centrifugation step the cap is screwed loosely onto the column 1 Sample Preparation This step ensures that the protein solution is at the proper pH for column binding a b c Obtain protein sample If particulates are present clarify the sample through either filtration or centrifugation Determine the pH and volume of the protein sample A
15. hould be no liquid in the column If necessary spin an additional two minutes to dry 5 Protein Elution The Elution Buffer that is supplied is 10 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 It is recommended that upon elution the protein solution is neutralized immediately especially for proteins that are known to be sensitive to high pH It is therefore recommended that the Protein Neutralizer that is provided is pre added to the elution tube However this is not needed for the first elution For the second elution 0 35 mL of Protein Neutralizer is needed Note Please verify the pH of your first eluted protein sample and adjust with the Neutralizer if required a Add 350 uL Protein Neutralizer if desired not required for the 1st elution to a fresh 50 mL elution tube b Transfer the spin column with bound protein into the 50 mL elution tube from step 6a c Apply 4 mL of Elution Buffer C to the column and centrifuge for 2 minutes to elute the bound protein Note Approximately 90 of bound protein is recovered in the first two elutions If desired a second elution using Elution Buffer C may be carried out This should be collected into a different tube to which Protein Neutralizer is pre added to prevent dilution of the first elution Appendix 1 Optional Elution Buffers Proteins bound to Norgen s spin colum
16. mL Protein Neutralizer 2x4mL Maxi Spin Columns assembled with 4 collection tubes Elution tubes 50 mL 4 Product Insert 1 Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature Once opened the solutions should be stored at 4 C All the reagents should remain stable for at least 1 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only It is not intended for 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 e Swing Bucket centrifuge capable of spinning the 50 mL conical tubes pH indicator paper Micropipettors Isopropanol Milli Q water 100 mL sterile bottles Other elution buffers optional Notes prior to use e All centrifugation steps are carried out in a benchtop microcentrifuge at 1 000 x g except where noted Please check your microcentrifuge specifications to ensure proper speed Performance of the kit is not affected by temperature and thus the procedure may be performed at room temperature 4 C or on ice e Ensure that all solutions are at room temperature prior to use and that no precipitates have formed If necessary
17. ns Protein may have precipitated prior to loading onto the column If the pH of the protein solution is the same as the pl of the protein s precipitation may occur In this case adjust the pH of the sample to at least 1 pH unit lower than the pl of your protein Eluted protein is degraded Eluted protein was not neutralized Add 300 uL of Neutralizer to each 4 mL of eluted protein for all basic protein solutions Acidic proteins tend not to need the neutralization for the first elution but require it for the second or third Check the pH of the first elution if you are using an acidic protein Proteases may be present Use protease inhibitors during all steps of the Sample Preparation Bacterial contamination of protein solution Prepare the protein sample with 0 015 sodium azide The elution buffer already contains sodium azide Eluted protein was not neutralized quickly enough If eluted protein is not neutralized immediately degradation will occur We strongly recommend adding Neutralizer in order to lower pH 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 2014 Norgen Biotek Corp P117150 1 M14 11
18. ns 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 of the protein of interest Solutions not provided with the ProteoSpin Detergent Clean Up Maxi Kit may be utilized if they are more appropriate for your needs The table below lists optional elution buffers and their observed efficiency when BSA is used as a test protein Elution Buffers Approximate Protein Recovery 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 approximate pH 12 5 lt 70 100 mM sodium borate approximate pH 12 5 95 100 1 M Tris approximate pH 12 5 95 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 10 Troubleshooting Guide Problem Possible Cause Solution and Explanation f Check the centrifuge and ensure that it is capable of generating Centrifugation speed 1 000 x g Sufficie
19. nt centrifugal force is required to push the was too low nae liquid through the column Inadequate spin time Spin an additional minute or two to ensure the liquid has passed through the resin Protein Dilute the protein solution and adjust the pH to either 4 5 or 7 solution does Protein solution is too with the appropriate pH Binding Buffer Highly viscous not flow viscous materials due to high protein concentrations can slow down flow through the rate significantly column Cellular debris is Prior to the sample preparation step filter the sample with a present in the protein 0 45 uM filter or spin down insoluble materials Solid insoluble solution materials can cause severe clogging problems Protein solution is not Dissolve the sample in a larger amount of buffer Solid completely dissolved insoluble materials can cause clogging problems Initial v l me e Load at least 4 mL onto the column This volume ensures that sample apphedto the the entire bed is covered sufficientl column was too low y Ensure that the acidic protocol was used for acidic proteins and Incorrect procedure the basic protocol was used for basic proteins It is known that was used when basic proteins are bound with the acidic protocol elution Poor peptide is inefficient because the basic proteins are bound too tightly recovery Incorrect pH adjustment of sample Ensure that the pH of the starting protein sample is 4 5 for acidic proteins and 7 0 for basic protei
20. protein sample has been applied to the column d Discard any remaining flowthrough and reassemble the spin column with its collection tube 4 Column Wash a Apply 10 mL of Wash Solution C to the column and centrifuge for two minutes b Discard the flowthrough and reassemble the spin column with its collection tube c Add 10 mL of Wash Solution C to the column and centrifuge for two minutes d Inspect the column and ensure that the liquid has passed through into the collection tube There should be no liquid in the column If necessary spin an additional two minutes to dry 5 Protein Elution The Elution Buffer that is supplied is 10 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 It is recommended that upon elution the protein solution is neutralized immediately especially for proteins that are known to be sensitive to high pH It is therefore recommended that the Protein Neutralizer that is provided is pre added to the elution tube However this is not needed for the first elution For the second elution 0 35 mL of Protein Neutralizer is needed Note Please verify the pH of your first eluted protein sample and adjust with the Neutralizer if required a Add 350 uL Protein Neutralizer if desired not required for the 1st elution to a fresh 50 mL elution tube b Transfer the spin column with bound protein into t
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