Purification of Strep-tagged Proteins Using PureCube HiCap StrepTactin Agarose

This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of Strep-tagged proteins under native conditions using PureCube HiCap StrepTactin Agarose. Reagent amounts given apply to 200 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). If other culture volumes are processed or protein expression is higher or lower, reagent volumes may need to be adjusted. In this protocol cell lysis is done using lysozyme because it is an inexpensive and efficient method for cells that have been frozen. However, lysis methods based on physical disruption (e.g., sonication or homogenization) or detergents (e.g., CHAPS) can also be used.

Buffer compositions - Protein purification protocol

Lysis Buffer, 50 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

TRIS base, pH 8.0	100 mM	121.14	1 M	60.57 g/ 500 mL	5 mL
NaCl	150 mM	58.44	5 M	146.1 g/500 mL	1.5 mL
Protease inhibitor	1x	-	follow supplier‘s instructions
Lysozyme	1 mg/ml	-	-	-	50 mg
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. Add protease inhibitor and lysozyme directly before use.

Wash Buffer, 100 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

TRIS base, pH 8.0	100 mM	121.14	1 M	60.57 g/ 500 mL	10 mL
NaCl	150 mM	58.44	5 M	146.1 g/500 mL	3 mL
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0.

Elution Buffer, 10 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

TRIS base, pH 8.0	100 mM	121.14	1 M	60.57 g/ 500 mL	1 mL
NaCl	150 mM	58.44	5 M	146.1 g/500 mL	300 µL
Desthiobiotin	2.5 mM	214.26	25 mM	53 mg/10 mL	1 mL
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. Add desthiobiotin directly before use.

5X SDS Page Buffer, 10 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

Tris-HCl, pH 6.8–7.0	300 mM	121,14	1 M	121,14 g/ 1 L	3 mL
Glycerol	50% (v/v)	-	100% (v/v)	-	5 mL
SDS	5% (w/v))	-	-	-	0.5 g
Bromophenol blue	0.05% (w/v)	-	4%	-	125 μL
DTT	250 mM	154.25	1 M	1.54 g/ 10 mL	125 μL/aliquot
Instructions: Make sure to prepare a 1 M Tris-HCl stock by dissolving Tris base in 500 mL deionized water, adding HCl to a pH of 6.8–7.0, and adding water to a final volume of 1 L. For the SDS-PAGE Buffer, mix all components listed except DTT and add water to a total of 10 mL. Freeze 20 aliquots (375 µL each) at –20˚C. Before use, add DTT to the needed single aliquots.


A.	Protein purification protocol
	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. Resuspend the cell pellet in 10 mL Lysis Buffer, and pour it into a 50 mL conical centrifuge 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. Incubate on an end-over-end shaker at 4°C for 1 h. . Centrifuge the lysate for 30 min at 10,000 x g and 4˚C. Carefully collect the supernatant without touching the pellet. Note: The supernatant contains the soluble proteins and is the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis. Resuspend the PureCube HiCap StrepTactin Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 500 µL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column. Add 3 mL Wash Buffer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant. Repeat this step twice. Add 10 mL cleared lysate to the equilibrated PureCube HiCap StrepTactin Agarose resin and incubate at 4˚C for 1 h on an end-over-end shaker. Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Wash Buffer to rinse the centrifuge tube and remove resin adhered to the wall. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets. Remove the bottom cap of the column and collect the flowthrough. This is the flow-through fraction. Wash the column with 1 mL Wash Buffer. Repeat the washing step at least 3 times. These are the wash fractions Elute the Strep-tagged protein 5 times using 250 µL Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields. 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. . Optional: Perform Western Blot using Strep Antibody.

Protein purification protocol

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.
Resuspend the cell pellet in 10 mL Lysis Buffer, and pour it into a 50 mL conical centrifuge 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.
Incubate on an end-over-end shaker at 4°C for 1 h.
. Centrifuge the lysate for 30 min at 10,000 x g and 4˚C. Carefully collect the supernatant without touching the pellet. Note: The supernatant contains the soluble proteins and is the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
Resuspend the PureCube HiCap StrepTactin Agarose by inverting the bottle until the suspension is homogeneous. Transfer 1 mL of the 50% suspension (corresponding to 500 µL bed volume) to a 15 mL conical centrifuge tube. Allow the resin to settle by gravity and remove the supernatant. Tip: Alternatively, resin equilibration can be performed directly in the disposable gravity flow column.
Add 3 mL Wash Buffer and gently resuspend the slurry to equilibrate the resin. Allow the resin to settle by gravity and remove the supernatant. Repeat this step twice.
Add 10 mL cleared lysate to the equilibrated PureCube HiCap StrepTactin Agarose resin and incubate at 4˚C for 1 h on an end-over-end shaker.
Transfer the binding suspension to a disposable gravity flow column with a capped bottom outlet. Use Wash Buffer to rinse the centrifuge tube and remove resin adhered to the wall. Tip: Alternatively, batch binding can be performed directly in a gravity flow column with closed bottom and top outlets.
Remove the bottom cap of the column and collect the flowthrough. This is the flow-through fraction.
Wash the column with 1 mL Wash Buffer. Repeat the washing step at least 3 times. These are the wash fractions
Elute the Strep-tagged protein 5 times using 250 µL Elution Buffer. Collect each eluate in a separate tube and determine the protein concentration of each fraction. These are the elution fractions. Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields.
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.
. Optional: Perform Western Blot using Strep Antibody.