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

Properties

Form

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

Storage instruction

2 to 8°C, do not freeze

Beads Diameter

90 µm

Binding Capacity

Approx. 3-5 mg Strep tag II protein/mL settled beads

Ligand

StrepTactin (∼5mg StrepTactin/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

1XPBS, containing 20% Ethanol

Applications

Protocols

SOLUTIONS
Equilibration/ Wash Buffer:
100 mM Tris-HCl,150 mM NaCl,1 mM EDTA,pH8.0

Elution Buffer:
Add 2.5 mM D-Desthiobiotin or D-biotin to the Equilibration Buffer

Note: D-biotin binds very tightly to Streptactin, and after high concentration NaOH cleaning, the binding capacity can only reach about 50% of the initial.

Regeneration Buffer:

1 M NaOH or 
Add 1 mM HABA to Equilibration Buffer

PROTOCOL

Equilibrate the column
1. Add 2 CV ( column bed volume) of Equilibration/ Wash Buffer

Apply the protein extract
2. Add 0.5 up to 10 CV cell extract 

Frozen cell extracts have to be centrifuged after thawing (14.000 rpm, 5 min, 4 °C) in order to 

remove any aggregates that may have formed. Apply the cleared extract to the column

 

Wash column
3. Wash column 5x with 1 CV of Equilibration/ Wash Buffer

Collect the wash fractions (1 CV each) and apply 2 µl of each subsequent wash fraction to an analytical 

SDS-gel (additionally apply 2 µl of the lysate and 2 µl of the flow through).

Elute protein
4. Add 6x of 0.5 CV Elution Buffer

Collect the eluate in 0.5 CV fractions. To get most of Strep-tag® fusion protein in one fraction, i.e. E2, 

only, add 0.8 CV (E1), then 1.4 CV (E2) and finally again 0.8 CV (E3). 20 µl samples of each fraction can 

be used for SDS-PAGE analysis.

Regenerate column
5. Wash the column 3x with 5 CV Regeneration Buffer

The color change from yellow to red indicates the regeneration process. The intensity of the red color 

is an indicator for the column activity status for the next round of purification.

Remove HABA and equilibrate

6. Remove Regeneration Buffer by adding 2x 4 CV Equilibration/ Wash Buffer (pH 8)

This step is essentially the same for Strep-Tactin Sepharose®, Macroprep® or Superflow® (high 

capacity). If the column is not completely discolored after the removal step please wash with

Buffer W at pH 10.5. Afterwards the columns have to be stored in Buffer W at pH 8.

Store column
7. Store the column at 4 °C overlaid with 2 ml Equilibration/ Wash Buffer (pH 8).