Strep-tag® II: WSHPQFEK Twin-Strep-Tag® (Strep-tag III): WSHPQFEKGGGSGGGSGGWSHPQFE

Membrane proteins can be purified after solubilization in a nonionic detergent. Concentrations of up to 2% of Triton® or TWEEN® can be present in the loaded sample.

No, this will prevent the protein from binding to the matrix. To remove biotin from the sample we recommend to add stoichiometric amounts of Avidin prior to using the kit. Avidin blocks the biotin but does not bind to the Strep-tag. Alternatively, dialysis could be conducted.

The sample may contain up to: Reducing Agents ≤ 50 mM β-mercaptoethanol Non-Ionic Detergents ≤ 2% Nonidet® P-40 ≤ 2% Triton® X-100 ≤ 2% Tween®-20 Ionic Detergents ≤ 0.1% SDS (Sodium-N-dodecyl sulfate) Denaturing Reagents ≤ 8 M Urea Chemicals and other Reagents ≤ 50 mM EDTA ≤ 10% (v/v) Ethanol ≤ 25% (v/v) Glycerol ≤ 250 mM Imidazole ≤ 1 M MgCl2 ≤ 5 M NaCl

For protein purification from E. coli lysates: 1. Harvest & pellet 10 ml of E. coli culture. 2. Resuspend in 1-2 ml of Lysis Buffer (e.g. 300 mM NaCl,1% NP-40 in PBS, 1 mg/ml Lysozyme, 1 x Protease Inhibitor, 15 U/ml Benzonase®). 3. Incubate for 30 minutes at room temperature. 4. Spin at ≥ 12,000 x g at 4°C for 5 minutes. 5. Use the supernatant for the Strep-Spin Protein Miniprep Kit™ protocol.

– Check your buffers for signs of contamination and check the pH of the buffers. – Increase centrifugation time and speed. Ensure that the Strep-Tactin® XT Superflow® drains completely after each spin (some older centrifuge models may require a longer centrifugation time). – If the problem persists, additional wash steps can be added to the purification protocol.

Smaller elution volumes are possible and may yield more concentrated protein, but the elution efficiency may be compromised.

It will work without the equilibration step, however, for optimal performance we recommend including it.

– The starting material contains Biotin. Free biotin binds to the Strep-Tactin® XT matrix and thus reduces the binding capacity for the desired protein. To remove biotin from the sample we recommend to add stoichiometric amounts of Avidin prior to using the kit. Avidin blocks the biotin but does not bind to the Strep-tag. Alternatively, dialysis could be conducted. – The Strep-tag may be rendered inaccessible due to protein folding. The protein can be purified at denaturing conditions. – The recombinant protein can become insoluble as a result of overexpression. The protein can be purified at denaturing conditions. – Starting material is too dilute. If the starting material contains very low levels of Strep-tagged protein, then it may require more than 800 µl of sample volume to purify enough protein. Simply repeat steps 5 and 6 until the desired volume has been loaded onto the column. – Please ensure not to use higher detergents volumes as mentioned in the Sample Preparation table. Using higher detergent amounts can reduce binding affinity.

Optimize expression conditions. Overexpression of proteins may result in formation of insoluble inclusion bodies inside cells. If a large band of overexpressed protein is visible after SDS-PAGE electrophoresis of whole cells, but the band is absent after SDS-PAGE electrophoresis of cleared cell lysates, this indicates that the protein may not be soluble and the expressed protein may form inclusion bodies. – Use Denaturing conditions. Insoluble proteins will not be purified using the provided buffers. It is, however, possible to purify such proteins at denaturing conditions in the presence of ≤ 8 M urea or ≤ 6 M guanidine hydrochloride. The protein native structure and thus enzyme activity is lost under such conditions, but may be restored by refolding the protein after purification.