The Strep-tag® purification system is based on the highly selective binding of engineered streptavidin, called Strep-Tactin, to Strep-tag II fusion proteins. PureCube StrepTactin MagBeads are ferrimagnetic agarose beads coupled to the Strep-Tactin® protein; a useful system for protein purification and pull-down experiments to determine protein-protein interactions. PureCube HiCap StrepTactin MagBeads are delivered as a 5% suspension. For detection of strep-tagged proteins in Western Blots specific antibodies and antibody conjugates
PureCube HiCap StrepTactin MagBeads provide:
- Binding capacity of up to 7 mg protein per ml settled beads
- purification of proteins expressed at low levels or from diluted solutions
- optimal conditions for pull-down experiments
- low unspecific binding
- HiCap StrepTactin Agarose also available
||Specific binding and purification of Strep-tagged®
||Affinity to Strep-tagged® proteins
|High binding capacity
||7 mg/mL settled beads
||Delivered as a 5 % suspension
|| 10% Ethanol; 5M NaCl. 0,1% SDS; 2% Tween; 25% glycerol
|A.||Protein purification protocol für native conditions:|
- Thaw the E. coli cell pellet on ice. 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 1 mL Lysis Buffer.
- Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
- Incubate for 30 min on ice.
- Centrifuge the lysate for 30 min at 10,000xg and 4˚C. Collect the supernatant. 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 MagBeads by vortexing. Transfer 200 μL of the 5% magnetic bead suspension into a conical microcentrifuge tube (or the volume adjusted to the expression level
- Add 1 mL Wash Buffer and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
- Repeat step 7 twice.
- Pipet 1 mL of the cleared lysate onto the equilibrated magnetic beads, and incubate the lysate-magnetic bead mixture at 4˚C for 1 h on an end-over-end shaker
- Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Note: This is the flow-through fraction
- Remove the tube from the magnet. Add 500 µL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant. Note: These are the wash fractions.
- Repeat step 11 twice
- Elute the Strep-tagged protein using 100 μL Elution Buffer.
- Repeat step 13 five times. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction. Note: These are the elution fractions.Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields
- Optional: 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.
StrepTactin® and Strep-tag® are trademarks of IBA GmbH. PureCube StrepTactin protein is manufactured by IBA GmbH under German Patent Application No. 42 37 113.9 entitled "Fusion peptides with binding activity for streptavidin"