1. Overview

Strep-Tag system is a novel protein purification system that mimics the streptavidin-biotin system, and Strep-Tactin has at least 10 times stronger affinity for Strep-Tag II than streptavidin, and the separation and purification conditions are mild, so the protein can be separated and purified under physiological conditions. In addition, compared with other tags, Strep-Tag II is a small tag of 8 amino acids (WSHPQFEK), which is only about 1 kDa due to the small tag, which does not affect the structure and function of the protein after fusion. These mild purification parameters can preserve the biological activity of the protein and produce more than 99% purity after only one step extraction. Magarose-Strep-Tactin magnetic beads were covalently coupled to the surface of superparamagnetic agarose magnetic beads by a special protein coupling process, and a new functional material for the efficient and rapid separation and purification of Strep-tag II protein was prepared, and a protein purification platform with extraction speed, extraction amount and purity was realized and built.

2. Product characteristics

Product Name	Magarose- Strep-Tactin
Item number	PMAG003
Magnetic bead concentration	25% (v/v)  in 1×PBS (0.1% Tween-20+0.05% NaN3)
ligand density	~6 mg Strep-Tactin /ml beads
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	~7 mg Strep-tag II protein/ml beads
Save	Store at 2~8°C for one year

Note: The amount of magnetoglobin binding is related to the characteristics of the target protein, and is only used as a reference value.

3. How to use

1. Scope of application

Can be used for isolation and purification of Strep-Tag II-tagged proteins from any expression system, including baculovirus, mammalian cells, yeast, and bacteria.

2. Operation process

The binding performance of the target protein to the magnetic beads will directly affect the purification efficiency of the target protein, and the preparation of various buffers will also affect the recovery and purity of the target protein to a certain extent. The following provides a more widely used Strep-Tag II protein purification process, which users can refer to or design and optimize their own protein purification process according to their own protein characteristics.

2.1 Buffer solution preparation

Binding/Washing Buffer ：10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0；

Elution Buffer：2.5 mM desthiobiotin in Binding Buffer；

Regeneration Buffer: 0.5 M NaOH or 1 mM HABA in Binding Buffer；

2.2 Sample processing

This User Manual provides the following three sample handling methods:

(1) Intracellular expression proteins such as Escherichia coli and yeast: the expressing cells are diluted with an appropriate amount of Binding Buffer and protease inhibitors (such as PMSF with a final concentration of 1 mM); Ice bath ultrasound lyses of cells, which is a crude protein sample. If the sample is too viscous, an appropriate amount of nuclease can be added to the crude sample as needed and placed on ice for 30 minutes to degrade the nucleic acid. In addition, if the target protein content is low, it is recommended to centrifugate the crude protein sample.

(2) Extracellular expression protein: take the extracellular expression supernatant and dilute the equilibrium with the same amount of Binding Buffer, which is the crude protein sample.

(3) Intracellular expression protein of animal cells: take an appropriate amount of animal cells, wash them once with an appropriate amount of PBS, and discard the supernatant; Resuspend with a Binding Buffer containing an appropriate amount of 1% (v/v) Triton X-100 or 1% (v/v) NP-40; Add a protease inhibitor (e.g., PMSF at a final concentration of 1 mM); Place on ice for 10 minutes to get a crude protein sample.

2.3 Magnetic bead pretreatment

In general, the magnetic bead dosage is calculated by the user based on the target protein yield and magnetic bead load. For example, using Escherichia coli to express a target protein, 250 mL of fermentation broth to harvest 1 g of wet weight bacteria, the yield of the target protein is estimated to be ~7 mg through pre-experiments, and the user needs to take 4 mL of 25% magnetic bead suspension for the purification of the target protein. The following is an example to illustrate in detail:

(1) Place the magnetic beads on a vortex mixer and mix thoroughly, and use a pipette to take 10 mL of magnetic bead suspension in a centrifuge tube;

(2) Place the centrifuge tube on the magnetic separator and remove the supernatant;

(3) Add 5~10 mL of Binding Buffer/Washing Buffer to the centrifuge tube containing the magnetic beads, close the lid tightly, and vortex for 15 s to resuspend the magnetic beads. Place the centrifuge tube on a magnetic separator, magnetically separate, remove the supernatant, and repeat the wash twice.

(Note: In the process of magnetic separation, in order to reduce the loss during the use of magnetic beads, after the solution becomes clear, close the centrifuge tube lid tightly, keep the centrifuge tube still on the magnetic separator, hold the magnetic separator and the centrifuge tube up and down several times, wash the remaining magnetic beads on the centrifuge tube with a clarifying solution, and let it stand for a while to make the solution clear again; The same applies below. ）

2.4 The target protein binds to the magnetic beads

(1) Resuspend 1 g of wet weight bacteria with a 10 mL Binding Buffer, crush and lyse it, which is the crude protein sample;

(2) Transfer the crude protein sample to a centrifuge tube containing pretreated magnetic beads and close the tube cap tightly;

(3) Place the centrifuge tube in a vortex mixer for 15 s, then place it on a vertical mixer and mix vertically at room temperature for 30 min (if necessary, rotate and mix at a low temperature of 2~8°C for about 1 h to prevent degradation of the target protein);

(4) Place the centrifuge tube on the magnetic separator for magnetic separation, and remove the supernatant into a new centrifuge tube for subsequent testing. Remove the centrifuge tube from the magnetic separator for the next washing step.

2.5 Magnetic bead washing

(1) Add 5~10 mL Washing Buffer to the magnetic beads in step 4, swirl and mix for 2 minutes, magnetically separate, and remove the washing solution into a new centrifuge tube for sampling and testing.

(2) Continue to add 5~10 mL Washing Buffer to the above magnetic beads, swirl and mix for 2 minutes, re-suspend the beads, transfer the magnetic bead suspension to a new centrifuge tube, and avoid contamination of the target protein by non-specific adsorbed proteins on the wall of the original centrifuge tube; Magnetically separate, remove the supernatant from the collection tube for sampling and testing;

2.6 Eluting of the target protein

(1) Add 2~5 mL Elution Buffer (the user changes the elution volume to adjust the target protein concentration) into the centrifuge tube, close the centrifuge tube cap tightly, place the centrifuge tube on the vertical mixer, and mix vertically at room temperature for 10 minutes; Magnetic separation, collect the eluent into a new centrifuge tube, that is, the purified target protein sample;

(2) If needed, repeat the above step 1 time to collect the sample into a new centrifuge tube to detect whether the target protein is completely eluted.

2.7 Magnetic bead regeneration and preservation

(1) NaOH regeneration: The magnetic beads after eluting the target protein are washed in the following order: 5~10 mL purified water washed 3 times, 5~10 mL 0.5M NaOH washed 3 times, 5~10 mL purified water washed to neutral, and finally 10 mL of preservation solution was added, and the magnetic beads were placed in an environment of 2~8°C for storage.

(2) HABA regeneration: the magnetic beads after eluting the target protein with desulfiobiotin can also be regenerated with HABA buffer, adding 5~10 mL of 1mM HABA to wash the beads 5 times, then washing the beads to their own color with Binding Buffer, washing them for 5 minutes each time, and finally adding 4 mL of preservation solution, and placing the magnetic beads at 2~8°C for storage.

3. Optimization of protein purification processes

The above procedure is suitable for the purification of most Strep-Tag II tagged proteins, and depending on the binding performance of the target protein to the magnetic beads, users can optimize the purification process in the following aspects to improve the recovery and purity of the target protein.

3.1 Reference method to improve the recovery of target proteins

(1) Prolong the incubation time of protein solution and magnetic beads;

(2) Add appropriate protease inhibitors to prevent the degradation of the target protein;

(3) increase the amount of magnetic beads;

(4) prolonging the elution time or increasing the number of elutions;

3.2 Reference method for improving the purity of the protein of interest

(1) Add appropriate protease inhibitors during purification to prevent degradation of the target protein;

(2) Extend the washing time and increase the number of washes;