The Strep-tag® purification system is based on the highly selective binding of engineered streptavidin, called Strep-Tactin, to Strep-tag II fusion proteins. Cube Biotech offers a high-quality affinity resin for strep-tagged proteins, with maximum binding capacity. PureCube HiCap StrepTactin Agarose is delivered as a 50% suspension. For purification from cell culture supernatants or for pull-down experiments, PureCube HiCap StrepTactin® MagBeads are recommended. For detection of strep-tagged proteins in Western Blots, specific antibodies and antibody conjugates are available.
PureCube HiCap StrepTactin Agarose provides:
- Binding capacity of up to 5 mg protein per mL resin
- Stable in the presence of a wide range of additives
- High-quality performance for batch or gravity flow purification
- Also available as MagBeads and prepacked FPLC columns
||Specific binding and purification of 6x his-tagged proteins
||Affinity to His-tagged proteins
||2% Tween 20, 2% Triton X-100, 2% IGEPAL® 630/Nonidet P40, n-Octyl-beta-D-glucopyranoside, 0,2% n-Nonyl-beta-D-gluyopyranoside, 0,35% N-Decyl-beta-D-maltoside, 2% Lauryl-sacrosine, 0,1% SDS, 0,3% CHAPS, 1M Guanidine Hcl, 1mM PMSF, 10% Ethanol, 5M NaCl, 2M (NH4)SO4, 1M CaCl2, 25% Glycerol
||Delivered as a 50 % suspension
||100 μm, to obtain flow rates up to 6 mL/min
- 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
- Disposable gravity flow columns with capped bottom outlet, 2 ml
- pH meter
- Optional: 15 mL conical propylene tubes (e.g. Falcon)
- End-over-end shaker
- SDS-PAGE buffers, reagents and equipment Optional: Western Blot reagents and equipment
|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.
StrepTactin® and Strep-tag® are trademarks of IBA GmbH. PureCube StrepTactin Agarose is manufactured by IBA GmbH under German Patent Application No. 42 37 113.9 entitled "Fusion peptides with binding activity for streptavidin".