Batch Spin Columns


Spin Columns
Empty 1-step batch columns are perfectly suited for batch purifications. They can be filled with your affinity resin of choice, and allow for binding, washing, and elution in one single spin column. Designed for small to mid-scale protein purification, the PureCube 1-step batch spin columns save time and pipetting steps. Featuring SelfSeal membrane technology, the column retains resin and sample in a chamber for batch incubation. By centrifugation, the membrane pores dilate and the filtered eluate gathers in the collection chamber of the column. PureCube 1-step batch spin columns are available in two sizes: Mini for expression trials, small-scale screening, and other small-volume purification needs; or Midi Plus for volumes of up to 20 mL.

1-step batch Columns are useful for:


Features

Feature PureCube 1-step batch Mini PureCube 1-step batch Midi Plus
Usage Specific binding and purification of proteins
Specifity Depending on the affinity of the used agarose resin
Binding capacity Also depending on the used agarose
Body material Polypropylene
Filter pore size 0.1-0.2µm low binding PVDF
Max. volume 600 µL (mini) 20 mL (midi)
Max. g force 12,000-14,000 x g (45-degree fixed angle rotor) 1,500-2,000 x g (swing-bucket rotor)
Min. g force 2,500 x g for 1 min 100-200 x g for 1-10 min
Min. g force 0.5-1 min at 12-14,000 x g 5-10 min at 400 x g
Required equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Microcentrifuge with rotor suitable for 2 mL centrifuge tubes (min 10,000 xg)
  • 2 mL centrifuge tubes
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  •  pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • 50 mL conical tubes (e.g. Falcon)
  •  pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment

PureCube 1-step Mini_250X250_RGB72
632xx PureCube 1-step Midi_insert_on_side_450X450_RGB72
Fig.1: PureCube 1-step batch Mini (left) and Midi Plus columns.

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
These protocols are examples for the use of our PureCube 1-step columns in combination with our His tag affinity agarose resins.
   
A.Protocol for purification under native conditions: MiniColumns
 
 
  1. Thaw the E. coli cell pellets corresponding to 10 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at –20˚C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 1 mL Lysis Buffer and pour it into a 2 mL microcentrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption. Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer. Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein. Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1–5 mM.
  4. Centrifuge the lysate at 10.000 x g for 30 min and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 200 µL of the 50% suspension (corresponding to 100 µL bed volume) into the batch incubation chamber of the spin column barrel. Close the chamber and spin the resin at 10.000-14.000 x g for 20 sec.
  6. Add 600 µL of Lysis Buffer and centrifuge again at 10.000- 14.000 x g for 20 sec.
  7. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  8. Immediately before loading, filter the cleared lysate prepared in step 4 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  9. Empty the 2 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  10. Load the lysate filtered in step 8. The maximum loading volume is 600 µL. Invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  11. Centrifuge at at 10.000-14.000 x g for 20 sec, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction.
  12. Wash twice with 600 µL each of Wash Buffer. These are the wash fractions.
  13. Replace the 2 mL microcentrifuge tube with a fresh one, and elute the His-tagged protein by adding 50-600 µL Elution Buffer and centrifuging for 20 sec at 10.000-14.000 x g.
  14. Repeat the previous four times, for a total of five elutions. Collect each elution fraction separately. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  15. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  16. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  17. Optional: Perform Western Blot experiment using PentaHis Antibody.
 B.Protocol for purification under denaturing conditions: MiniColumns
 
 
  1. Thaw the E. coli cell pellets corresponding to 10 mL bacterial culture on ice for 15 min. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Read: “About Denaturation”. Nucleic acids can also be sheared by passing the lysate 10 times through a fine-gauge needle.
  2. Resuspend the cell pellet in 1 mL Denaturing Lysis Buffer and pour it into a 2 mL microcentrifuge tube.
  3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein. Optional: Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  4. Centrifuge the lysate at 10.000 x g for 30 min and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 200 µL of the 50% suspension (corresponding to 100 µL bed volume) into the batch incubation chamber of the spin column barrel. Close the chamber and spin the resin at 10.000-14.000 x g for 20 sec.
  6. Add 600 µL of Denaturing Lysis Buffer and centrifuge again at 10.000-14.000 x g for 20 sec.
  7. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  8. Immediately before loading, filter the cleared lysate prepared in step 4 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  9. Empty the 2 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  10. Load the lysate filtered in step 8. The maximum loading volume is 600 µL. Invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  11. Centrifuge at at 10.000-14.000 x g for 20 sec, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction.
  12. Wash twice with 600 µL each of Denaturing Wash Buffer. These are the wash fractions.
  13. Replace the 2 mL microcentrifuge tube with a fresh one. Elute the His-tagged protein by adding 50-600 µL Denaturing Elution Buffer and centrifuging for 20 sec at 10.000 - 14.000xg. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  14. Repeat step 13 four times, for a total of five elutions. Collect each elution fraction separately.
  15. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  16. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  17. Optional: Perform Western Blot experiment using PentaHis Antibody.
 C.Protocol for purification under native conditions: Midi Plus Columns
 
 
  1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at –20˚C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 10 mL Lysis Buffer supplemented with 1 mg/ml lysozyme, and pour it into a 50 mL centrifuge tube. Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer. Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  3. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption. Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1–5 mM.
  4. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein.
  5. Centrifuge the lysate for 30 min at 10,000 x g and 2–8˚C. Carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 500 µL bed volume) into the batch incubation chamber of the spin column barrel. Use the clear spin push cap to close the chamber and spin the resin at 400 x g for 5 min.
  7. Add 15 mL of Lysis Buffer and centrifuge again for 5 min at 400 x g.
  8. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  9. Immediately before loading, filter the cleared lysate prepared in step 5 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  10. Empty the 50 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  11. Load the lysate filtered in step 8. Tightly screw the yellow batch incubation cap and invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  12. After batch incubation, replace the yellow cap with the clear spin push cap. Centrifuge at 400 x g for 5 min, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction
  13. Wash twice with 15 mL each of Wash Buffer. These are the wash fractions.
  14. Replace the 50 mL conical centrifuge tube, and elute the Histagged protein by adding 1 mL Elution Buffer.
  15. Repeat step 14 four times, for a total of five elutions. Collect each elution fraction separately. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  16. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  17. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  18. Optional: Perform Western Blot experiment using PentaHis Antibody.
 D.Protocol for purification under denaturing conditions: Midi Plus Columns
 
 
  1. Thaw the E. coli cell pellet on ice. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Read: “About Denaturation”. Nucleic acids can also be sheared by passing the lysate 10 times through a fine-gauge needle.
  2. Resuspend the cell pellet in 10 mL Denaturing Lysis Buffer. Optional: Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  3. Incubate at room temperature for 30 min on an end-overend shaker.
  4. Centrifuge the lysate for 30 min at room temperature and 10,000 x g. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 500 µL bed volume) into the batch incubation chamber of the spin column barrel. Use the clear spin push cap to close the chamber and spin the resin at 400 x g for 5 min.
  6. Add 15 mL of Denaturing Lysis Buffer and centrifuge again for 5 min at 400 x g.
  7. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  8. Immediately before loading, filter the cleared lysate prepared in step 4 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  9. Empty the 50 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  10. Load the lysate filtered in step 8. Tightly screw the yellow batch incubation cap and invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  11. After batch incubation, replace the yellow cap with the clear spin push cap. Centrifuge at 400 x g for 5 min, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction.
  12. Wash twice with 15 mL each of Wash Buffer. These are the wash fractions.
  13. . Replace the 50 mL conical centrifuge tube, and elute the Histagged protein by adding 1 mL Denaturing Elution Buffer. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  14. Repeat step 13 four times, for a total of five elutions. Collect each elution fraction separately. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  15. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  16. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  17. Optional: Perform Western Blot experiment using PentaHis Antibody.
Spin Columns
Empty 1-step batch columns are perfectly suited for batch purifications. They can be filled with your affinity resin of choice, and allow for binding, washing, and elution in one single spin column. Designed for small to mid-scale protein purification, the PureCube 1-step batch spin columns save time and pipetting steps. Featuring SelfSeal membrane technology, the column retains resin and sample in a chamber for batch incubation. By centrifugation, the membrane pores dilate and the filtered eluate gathers in the collection chamber of the column. PureCube 1-step batch spin columns are available in two sizes: Mini for expression trials, small-scale screening, and other small-volume purification needs; or Midi Plus for volumes of up to 20 mL.

1-step batch Columns are useful for:


Features

Feature PureCube 1-step batch Mini PureCube 1-step batch Midi Plus
Usage Specific binding and purification of proteins
Specifity Depending on the affinity of the used agarose resin
Binding capacity Also depending on the used agarose
Body material Polypropylene
Filter pore size 0.1-0.2µm low binding PVDF
Max. volume 600 µL (mini) 20 mL (midi)
Max. g force 12,000-14,000 x g (45-degree fixed angle rotor) 1,500-2,000 x g (swing-bucket rotor)
Min. g force 2,500 x g for 1 min 100-200 x g for 1-10 min
Min. g force 0.5-1 min at 12-14,000 x g 5-10 min at 400 x g
Required equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Microcentrifuge with rotor suitable for 2 mL centrifuge tubes (min 10,000 xg)
  • 2 mL centrifuge tubes
  • Micropipettor and Micropipetting tips
  • Disposable gravity flow columns with capped bottom outlet, 2 ml
  •  pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment
 
  • Lysis Buffer
  • Wash Buffer
  • Elution Buffer
  • Ice bath
  • Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
  • 50 mL centrifuge tube
  • Micropipettor and Micropipetting tips
  • 50 mL conical tubes (e.g. Falcon)
  •  pH meter
  • End-over-end shaker
  • SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment

PureCube 1-step Mini_250X250_RGB72
632xx PureCube 1-step Midi_insert_on_side_450X450_RGB72
Fig.1: PureCube 1-step batch Mini (left) and Midi Plus columns.

Applications

All protocols and buffer compositions are also avaible as PDF-Files on the Protocols & Datasheets page.
These protocols are examples for the use of our PureCube 1-step columns in combination with our His tag affinity agarose resins.
   
A.Protocol for purification under native conditions: MiniColumns
 
 
  1. Thaw the E. coli cell pellets corresponding to 10 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at –20˚C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 1 mL Lysis Buffer and pour it into a 2 mL microcentrifuge tube. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption. Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer. Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein. Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1–5 mM.
  4. Centrifuge the lysate at 10.000 x g for 30 min and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 200 µL of the 50% suspension (corresponding to 100 µL bed volume) into the batch incubation chamber of the spin column barrel. Close the chamber and spin the resin at 10.000-14.000 x g for 20 sec.
  6. Add 600 µL of Lysis Buffer and centrifuge again at 10.000- 14.000 x g for 20 sec.
  7. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  8. Immediately before loading, filter the cleared lysate prepared in step 4 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  9. Empty the 2 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  10. Load the lysate filtered in step 8. The maximum loading volume is 600 µL. Invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  11. Centrifuge at at 10.000-14.000 x g for 20 sec, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction.
  12. Wash twice with 600 µL each of Wash Buffer. These are the wash fractions.
  13. Replace the 2 mL microcentrifuge tube with a fresh one, and elute the His-tagged protein by adding 50-600 µL Elution Buffer and centrifuging for 20 sec at 10.000-14.000 x g.
  14. Repeat the previous four times, for a total of five elutions. Collect each elution fraction separately. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  15. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  16. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  17. Optional: Perform Western Blot experiment using PentaHis Antibody.
 B.Protocol for purification under denaturing conditions: MiniColumns
 
 
  1. Thaw the E. coli cell pellets corresponding to 10 mL bacterial culture on ice for 15 min. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Read: “About Denaturation”. Nucleic acids can also be sheared by passing the lysate 10 times through a fine-gauge needle.
  2. Resuspend the cell pellet in 1 mL Denaturing Lysis Buffer and pour it into a 2 mL microcentrifuge tube.
  3. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein. Optional: Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  4. Centrifuge the lysate at 10.000 x g for 30 min and carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 200 µL of the 50% suspension (corresponding to 100 µL bed volume) into the batch incubation chamber of the spin column barrel. Close the chamber and spin the resin at 10.000-14.000 x g for 20 sec.
  6. Add 600 µL of Denaturing Lysis Buffer and centrifuge again at 10.000-14.000 x g for 20 sec.
  7. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  8. Immediately before loading, filter the cleared lysate prepared in step 4 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  9. Empty the 2 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  10. Load the lysate filtered in step 8. The maximum loading volume is 600 µL. Invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  11. Centrifuge at at 10.000-14.000 x g for 20 sec, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction.
  12. Wash twice with 600 µL each of Denaturing Wash Buffer. These are the wash fractions.
  13. Replace the 2 mL microcentrifuge tube with a fresh one. Elute the His-tagged protein by adding 50-600 µL Denaturing Elution Buffer and centrifuging for 20 sec at 10.000 - 14.000xg. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  14. Repeat step 13 four times, for a total of five elutions. Collect each elution fraction separately.
  15. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  16. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  17. Optional: Perform Western Blot experiment using PentaHis Antibody.
 C.Protocol for purification under native conditions: Midi Plus Columns
 
 
  1. Thaw the E. coli cell pellets corresponding to 200 mL bacterial culture on ice for 15 min. Optional: Freezing the cell pellet at –20˚C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 10 mL Lysis Buffer supplemented with 1 mg/ml lysozyme, and pour it into a 50 mL centrifuge tube. Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer. Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  3. If the solution is very viscous, add 3 units Benzonase® per mL E.coli culture volume to the lysis buffer. Alternatively or additionally, sonicate the lysate to improve cell disruption. Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1–5 mM.
  4. Incubate on an end-over-end shaker at room temperature for 30 min, or at 4°C for 1 h, depending on the temperature stability of the protein.
  5. Centrifuge the lysate for 30 min at 10,000 x g and 2–8˚C. Carefully collect the supernatant without touching the pellet. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 500 µL bed volume) into the batch incubation chamber of the spin column barrel. Use the clear spin push cap to close the chamber and spin the resin at 400 x g for 5 min.
  7. Add 15 mL of Lysis Buffer and centrifuge again for 5 min at 400 x g.
  8. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  9. Immediately before loading, filter the cleared lysate prepared in step 5 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  10. Empty the 50 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  11. Load the lysate filtered in step 8. Tightly screw the yellow batch incubation cap and invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  12. After batch incubation, replace the yellow cap with the clear spin push cap. Centrifuge at 400 x g for 5 min, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction
  13. Wash twice with 15 mL each of Wash Buffer. These are the wash fractions.
  14. Replace the 50 mL conical centrifuge tube, and elute the Histagged protein by adding 1 mL Elution Buffer.
  15. Repeat step 14 four times, for a total of five elutions. Collect each elution fraction separately. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  16. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  17. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  18. Optional: Perform Western Blot experiment using PentaHis Antibody.
 D.Protocol for purification under denaturing conditions: Midi Plus Columns
 
 
  1. Thaw the E. coli cell pellet on ice. Optional: Benzonase® can be added to the lysate to reduce viscosity caused by nucleic acids (3 U/mL bacterial culture). Read: “About Denaturation”. Nucleic acids can also be sheared by passing the lysate 10 times through a fine-gauge needle.
  2. Resuspend the cell pellet in 10 mL Denaturing Lysis Buffer. Optional: Up to 20 mM EDTA and 20 mM DTT can be used with INDIGO Ni-Agarose; NTA and IDA agaroses tolerate up to 1 mM EDTA and 10 mM DTT.
  3. Incubate at room temperature for 30 min on an end-overend shaker.
  4. Centrifuge the lysate for 30 min at room temperature and 10,000 x g. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube His Affinity Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 500 µL bed volume) into the batch incubation chamber of the spin column barrel. Use the clear spin push cap to close the chamber and spin the resin at 400 x g for 5 min.
  6. Add 15 mL of Denaturing Lysis Buffer and centrifuge again for 5 min at 400 x g.
  7. Repeat the step to completely remove any residual ethanol that might interfere with protein binding to the affinity resin.
  8. Immediately before loading, filter the cleared lysate prepared in step 4 through a 0.2 µm filter (e.g. syringe filter) to remove any solid material that might clog the column. Note: It is critical to perform this filter step immediately before loading the column.
  9. Empty the 50 mL centrifuge tube and place the spin column barrel containing the equilibrated purification resin back into it.
  10. Load the lysate filtered in step 8. Tightly screw the yellow batch incubation cap and invert 2-3 times to mix sample and resin. Incubate at 4°C for 1 h on an end-over-end shaker.
  11. After batch incubation, replace the yellow cap with the clear spin push cap. Centrifuge at 400 x g for 5 min, or until the lysate has completely passed through, and collect the flowthrough. This is the flow-through fraction.
  12. Wash twice with 15 mL each of Wash Buffer. These are the wash fractions.
  13. . Replace the 50 mL conical centrifuge tube, and elute the Histagged protein by adding 1 mL Denaturing Elution Buffer. Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
  14. Repeat step 13 four times, for a total of five elutions. Collect each elution fraction separately. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
  15. Determine the protein concentration of the elution fractions with Bradford assay, using BSA as protein standard.
  16. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  17. Optional: Perform Western Blot experiment using PentaHis Antibody.
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