Streptactin-XT Agarose Resin 6FF for Strep-tag II Fusion Protein Purification

Properties

Form

10 mL 50% slurry (5 mL settled beads); 20 mL 50% slurry (10 mL settled beads); 50 mL 50% slurry (25 mL settled beads);

Storage instruction

2 to 8°C, do not freeze

Structure Image

Beads Diameter

90 µm

Binding Capacity

Approx. 6mg Strep tag II protein/mL settled beads

Ligand

StrepTactin-XT (∼5mg StrepTactin-XT/mL settled beads)

Matrix

Agarose Beads 6FF beads, highly crosslinked 6% agarose

Maximum pressure

0.3 MPa, 3 bar

Chemical stability

stable in all commonly used buffers, reducing agents, denaturants, and detergents

Storage buffer

1*PBS, containing 20% Ethanol

Applications

Highlights

Advantages of Steptactin-XT Agarose Resin 6FF

 

Streptactin-XT and Streptactin are two products used for protein purification, both of which can be used to bind to fusion proteins with the Strep-tag II tag. Streptactin-XT Agarose Beads is an upgraded product of the Streptactin series for protein purification, which has the following advantages:

 

1. Streptactin-XT can withstand different buffer conditions and additives, such as high salt, detergents, reducing agents, metal ions, and chelating agents. It can be used for protein purification under denaturing conditions. D-biotin below 50 μM in the sample will not affect the binding of the target protein to Streptactin-XT.

 

2. After binding to the target protein, this product can be reversibly eluted with an elution buffer for Streptactin-XT containing 50 mM D-biotin, eliminating the need for desthiobiotin and reducing reagent usage.

 

3. Streptactin-XT Agarose beads can be regenerated and used for more than 30 times with the regeneration buffer for Streptactin-XT without affecting the binding performance. If you need to increase the regeneration intensity, you can use a newly configured 10 mM NaOH. It is recommended to use it for regeneration within 5 times.

Work Flow Image

Protocols

Purification Protocol

1. Gravity Column packing

1). Based on the estimated total amount of Strep-tag II tag protein in the sample, calculate the loading volume of the Streptactin-XT beads and add the required volume of beads to the gravity column.

 

2). After the beads to the bottom of the gravity column, equilibrate the beads with at least 3 column beads volumes of distilled water, and then equilibrate the beads with 5 column beads volumes of Binding / Equilibration Buffer (BE buffer for Streptactin-XT).

 

2. Sample preparation 

1). Adjust the sample to the composition of the binding buffer. For example, dilute the sample with Binding / Equilibration Buffer or buffer exchange using a desalting column.

 

2). Pass the sample through a 0.22 μm or a 0.45 μm filter and/or centrifuge it immediately before applying it to the column. If the sample is too viscous, to prevent it from clogging the column dilute it with Binding / Equilibration Buffer, increase lysis treatment (sonication, homogenization), or add DNase/RNase to reduce the size of nucleic acid fragments.

 

3. Binding of Recombinant Strep tag II proteins 

1). Add the clear sample containing the Strep-tag II fusion protein to the equilibrated gravity column, keep the sample in the column for at least 2 minutes to ensure fully binding of the sample to the beads, and collect the flow-through at a flow rate of 0.5-1 mL per minute (the flow-through valve can accurately control the flow rate). Then add the flow-through to the column again and repeat the previous operation. Repeated loading can improve binding efficiency. Collect the final flow through liquid for use as a control for subsequent testing.

 

 

4. Wash of Recombinant Strep tag II proteins 

1). Wash with 5 column beads volumes of Wash Buffer to remove non-specific proteins, repeat twice. Until the A280 of the ef?uent becomes stable at a flow rate of 1 mL/minute, 

2). The components of the washing flow through liquid were collected for use as negative controls for subsequent testing.

Important Note:

If high background (excessive non-specific binding) is observed in subsequent experiments, the duration and number of washing steps can be increased.

 

5. Elution of Recombinant Strep tag II proteins 

1). Elute with 3-5 column beads volumes of Elution Buffer (containing D-biotin), collect in batches, collect one tube per column volume, and detect separately. This ensures that all bound target proteins are eluted, while also obtaining high purity and concentration of the protein.

 

Note: Desthiobiotin cannot be used to elute this product.

In case of difficulty in protein elution, the concentration of D-biotin in the Elution Buffer and the number of elution cycles can be appropriately increased.

 

6. After purification, the beads can be regenerated as follows:

1). Wash with 5 column beads volumes of Wash Buffer

2). Regenerate the beads with 5 column volumes of Regeneration buffer for Streptactin-XT, Remove immediately.

3). Wash the beads with 8 column volumes of Wash Buffer, repeat twice.

4). After regeneration and wash, the beads is stored in an equal volume of Wash buffer at 2-8°C.