How to improve your Strep-tag® protein purification results:
Use Strep-Tactin®XT for higher protein yields
When protein yield is an issue, we recommend to use Strep-Tactin®XT, which allows for improved binding capacity and total protein yield. The reason for this effect is its improved binding affinity.
Strep-Tactin® binds to Strep-tag®II with an affinity of 1 μM which is nearly 100-times higher compared to the Streptavidin:Strep-tag®II binding. Twin-Strep-tag® is a sequential arrangement of two Strep-tag®II sequences, which enables the same mild and rapid purification as Strep-tag®II but, in addition, has an increased affinity for Strep-Tactin® (low nM range). Further, the Twin-Strep-tag® allows efficient purification even in batch or of diluted proteins e.g. directly from cell culture supernatants.
The recently developed Strep-Tactin®XT (xtra tight) has a binding affinity in low pM ranges for Twin-Strep-tag® still maintaining binding reversibility and mild recovery of immobilized proteins. Hence, Strep-Tactin®XT enables highest protein purities under physiological conditions in combination with Twin-Strep-tag®. The protein yield increases almost 2-fold compared to purification via Strep-Tactin® independent of the chosen tag.
Use MagStrep "type3" XT beads
For purification in batch format we recommend to use Strep-Tactin®XT conjugated to magnetic beads instead of Sepharose or Superflow resin.
MagStrep “type3” XT beads are Strep-Tactin®XT coated ferri-magnetic spheres with the following characteristics:
- high binding capacity
- very low non-specific protein binding due to improved coating
- flexible elution conditions, under denaturing conditions by boiling in SDS gel loading buffer or under
native conditions with biotin
- high affinity for Strep-tag®II and Twin-Strep-tag® – due to new Strep-Tactin®XT
These characteristics lead to very good purification results and make the MagStrep “type3” XT beads a superior tool for small scale purifications and purification in batch format with high demands on protein purity.
MagStrep "type3" XT beads
|2-4090-002||MagStrep "type3" XT beads 5% suspension||
Get automated with Strep-Tactin®XT Superflow® cartridges
For automatd protein purification of Strep-tag®II and Twin-Strep-tag® fusion proteins use IBA's Strep-Tactin®XT Superflow® cartridges (also available: Strep-Tactin® Superflow® and Strep-Tactin® Superflow® high capacity cartridges) for Äkta, peristaltic pumps and other HPLC/FPLC systems.
Manual protein purification allows for easy set-up since no instrumentation such as pumps and valves are required. However, there is a drawback to manual protein purification: the process is time consuming and labor intensive.
Therefore, automated protein purification via Strep-Tactin®XT Superflow® cartridges are recommended when the following facts are an issue:
- On-line monitoring
- Time and labor savings
|2-4021-001||Strep-Tactin® Superflow®XT cartridge (with 10-32 connection for HPLC and Äkta)||1 x 1 ml|
|2-4022-001||Strep-Tactin® Superflow®XT cartridge (with 10-32 connection for HPLC and Äkta)||1 x 5 ml|
His-STREPPER - the His/Strep-tag®II Adapter - is a molecule comprising Strep-tag®II (SA-WSHPQFEK) conjugated with a nickel charged trisNTA. It tightly binds to His-tag and thereby modifies the His-tag fusion protein into a Strep-tag®II presenting fusion protein without the need for cloning.
His-STREPPER should be applied to the His-tag fusion protein present in the initial cell lysate or to the His-tag eluate (after complete removal of imidazole) if a higher protein purity is required.
The modified recombinant protein carrying the His-STREPPER binds to a Strep-Tactin®XT column and can be purified further according to the Strep-tag® protocol.
Elution of the Strep-tag®II protein can be performed either with imidazole (to elute the His-tag protein without the Adapter) or with biotin (to elute the Strep-tag® protein).
His-STREPPER offers the advantages of the Strep-tag® purification system, namely the high purity of the isolated proteins, without a time-consuming cloning process to His-tag users.
The trick is to stepwise add biotin to the buffer and measure the pH inbetween. Biotin is not soluble under acidic conditions, therefore the pH needs to be adjusted while adding biotin into the buffer.
Contamination of biotin is often a problem during mammalian expression, when the protein is secreted into the medium. Mammalian expression media contain high amounts of biotin (vitamin H). Biotin will block the column and the Strep-tag®II or Twin-Strep-tag® target protein cannot bind to the resin. Therefore it needs to be blocked before it can be applied to a Strep-Tactin® or Strep-Tactin®XT column. For this the BioLock solution (Biotin blocking solution) can be used. Per 70 µg of biotin add 1 ml of this solution. Further information can be found here.
Intrinsic biotin or biotinylated proteins are only abundant in very low concentrations and do not disturb the purification process or the purity of the target protein. To completely avoid those proteins small amounts of Avidin or BioLock can be added.
In general the interaction of Strep-tag®II and Twin-Strep-tag® to Strep-Tactin®XT is quite stable and not influenced by detergents or other additives. A list of so far tested reagents can be found here.