Isolates transiently and weakly interacting proteins
Suitable to identify several binding partners/co-factors at once
Positive control system included!
The new One-STrEPTM system is an extremely fast and efficient method to identify protein complexes. While other systems require tedious optimization because of high background or two successive purification steps, the One-STrEP system requires one STEP only to isolate intact protein complexes. In this system Strep-Tactin columns are used for fast protein purification under physiological conditions with low background enabling even the isolation of weakly associated binding partners. Details >>
The One-STrEP procedure
Cells are transfected with the plasmid coding for the bait protein fused to the One-STrEP-tag. After an appropriate expression period, cells are lysed and the bait together with bound proteins is isolated using a Strep-Tactin column. Proteins are resolved on a SDS-gel and specifically bound proteins e.g. proteins not appearing in the mock control can be identified. This is done by either excising the protein bands from the gel and subsequent mass spectrometric analysis or, if interaction partners are known or expected, by Western blot using specific antibodies.
One-STrEP Starter Kit
The Kit includes the plasmid pEXPR-IBA103* for bait cloning and expression as well as various Strep-Tactin columns and buffers for the isolation of protein complexes in different scales. Also, it contains the StrepMAB-Classic monoclonal antibody specific for One-STrEP-tag and conjugated to HRP for detection of the bait. As a control for the entire procedure, a control plasmid encoding a bait (PR65) and antibodies for a specifically binding prey (PP2Ac) are included as well. Both PR65 and PP2Ac are subunits of the phosphatase 2A protein complex, which is present in most mammalian cells. A positive detection of the prey in the eluate proves the successful isolation of an intact protein complex [Junttila et al., 2005, abstract].
Components of the One-STrEP Kits
Components
amount
One-STrEP
Starter Kit
One-STrEP
Follow up Kit
Gravity flow Strep-Tactin®
Superflow® columns
5 x 1 ml
X
X
Gravity flow Strep-Tactin®
Superflow® mini columns
One-STrEP-tag specific StrepMAB-Classic HRP conjugate; antibody for 15 Western blots
50 µl
X
X
Buffer L (lysis)
150 ml
X
X
Buffer W (washing)
120 ml
X
X
Buffer BE (elution)
25 ml
X
X
Control: vector with PR65 insert (bait)
5 µg
X
Control: PP2Ac affinity-purified goat polyclonal antibody (for 5 Western blots)
50 µl
X
Manual
1
X
X
* The pEXPR-IBA103 plasmid included in the One-STrEP-Kit is based on pEXPR-IBA3 but contains the new One-STrEP-tag instead of Strep-tag II. Junttila MR, Saarinen S, Schmidt T, Kast J, Westermarck J (2004) Single-step Strep-tag® purification for the isolation and identification of protein complexes from mammalian cells. Proteomics, 5, 1199-1203, 2005, abstract.
"One-tag tandem affinity purification" for protein:protein interaction studies
When using our One-STrEP system for protein:protein interaction analysis a second purification step is generally not required after Strep-Tactin affinity purification. For cases, in which nonetheless further purification is requested, we are offering new ready-to-use Gravity flow StrepMAB-Classic MacroPrep columns. These columns enable a second, completely independent purification step as both the resin (MacroPrep vs Superflow) and the affinity receptor (mAB vs Strep-Tactin) are substantially different. This "one-tag tandem affinity purification" keeps the extent of bait modification minimal and thus allows better complex formation.
Specifications of Gravity flow StrepMAB-Classic MacroPrep Columns
Support
MacroPrep® 50 (polymethacrylate)
Form
Pre-packed in 100 mM Tris/HCl pH 8.0, 1 mM EDTA, 150 mM NaCl
Strep-tag II fusion
protein binding activity
> 15 nmol/ml resin
Elution
Strep-tag II fusion proteins may be gently eluted by adding 0.5 mM free Strep-tag II peptide (Cat. No. 2-1018-002) to a physiological buffer or by applying a pH 3.5 buffer.
Stability
6 months after shipment
Storage
4 °C
Shipment
RT
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Columns used e.g. for sequential elution of prey proteins in protein:protein interaction studies
StrepMAB-Immo, a murine monoclonal antibody, binds SerAla-One-STrEP-tag (and SerAla- Strep-tag II) fusion proteins nearly irreversibly. Thus, these columns are designed for the stable and oriented immobilization of bait proteins. Analysis of interacting proteins is performed by sequential elution of prey proteins from the bait via Gravity flow StrepMAB-Immo MacroPrep Columns using adequate buffers. The bait proteins are presumed to stay bound under a wide range of buffer conditions.
Please note, that Gravity flow StrepMAB-Immo MacroPrep Columns are not recommended for the purification of One-STrEP-tag or Strep-tag II fusion proteins because elution is not possible under physiological conditions.
Specifications of Gravity flow StrepMAB-Immo MacroPrep Columns
Specifications of Gravity
flow StrepMAB-Immo
MacroPrep Columns
Nearly irreversible binding of C-terminally fused Strep-tag II with SerAla linker (rec. protein seq.- SerAla-TrpSerHisProGlnPheGluLys). The Strep-tag II will be linked automatically via the SerAla dipeptide to the C-terminus when the vectors for C-terminal fusion from IBA are used. Other linker sequences than SerAla lead to reduced affinity in the cases tested so far. The sequence SAWSHPQFEK fused to the N-terminal end of a recombinant protein has not been tested so far and, therefore, it is uncertain whether this equally mediates nearly irreversible binding.
Support
MacroPrep® 50 (polymethacrylate)
Form
Pre-packed in 100 mM Tris/HCl pH 8.0, 1 mM EDTA,
150 mM NaCl
Strep-tag II fusion
protein binding activity
> 15 nmol/ml resin
Stability
6 months after shipment
Storage
4 °C
Shipment
RT
Need buying or technical advice?
Contact a protein expert!
Prokaryotic One-STrEP vectors
