Strep-tag pull-down is a tag-based affinity purification approach that uses the high specificity of the Strep-tag II (or related tags) to streptavidin resins to capture a bait protein and its interacting partners under gentle conditions.
At Creative Proteomics, we provide a comprehensive Strep pull-down service to help you:
Key benefits
Strep pull-down is an affinity purification method in which a bait protein is fused to a Strep-tag (such as Strep-tag II or Twin-Strep-tag). The tagged bait is expressed in cells or in a recombinant system, then captured on streptavidin-based resins.
Under optimized lysis and washing conditions, the bait protein and its binding partners are captured on the resin. After non-specific proteins are removed, complexes are eluted under mild competitive conditions using desthiobiotin or biotin (depending on the resin system). This enables:
Because the interaction between Strep-tag and Strep-Tactin is strong and highly specific, Strep pull-down is particularly well-suited for clean enrichment of tagged bait proteins and their complexes, with good compatibility for downstream mass spectrometry.
Strep pull-down combined with LC–MS/MS is widely used in mechanism studies, target validation, and pathway mapping. Typical research questions include:
Our scientific team can help you translate your biological question into a suitable Strep pull-down–MS experiment, including controls and follow-up validation.
Clean, low-background enrichment
Highly specific Strep-tag / Strep-Tactin® binding helps remove non-specific proteins, making true interactors stand out clearly.
Gentle elution, intact complexes
Gentle competitive elution (Desthiobiotin/Biotin) preserves native protein–protein interactions and multi-subunit complexes.
Mass-spec–ready workflow
Compatible buffers and reagents feed directly into LC–MS/MS, reducing extra cleanup and sample loss.
From single target to full interactome
One enrichment workflow supports both WB validation of specific candidates and unbiased MS discovery.
Reproducible, publication-oriented design
Standardized protocols, built-in controls, and recommended replicates support consistent, paper-ready datasets.
| Feature | Strep Pull-down | Co-IP | GST Pull-down | RIME-MS | TAP-MS |
| Tag / Antibody requirement | Requires Strep-tag on bait; does not require a specific antibody for enrichment | Requires a high-quality antibody against the bait; strongly dependent on antibody performance | Requires GST-tag and recombinant expression; antibody optional for readout only | Requires antibodies (often nuclear receptors / TFs) and crosslinking | Requires specially designed multi-tag constructs (e.g., FLAG–Strep) |
| Interaction state | Native complexes under relatively gentle lysis and wash conditions | Native; weak interactions can be lost during stringent washing | Often semi-artificial or in vitro; may not fully reflect cellular context | Crosslinked chromatin-associated complexes | Enriches stable complexes with stringent multistep purification |
| Background level | Typically low to moderate, often cleaner than many antibody-based or metal-affinity methods | Highly variable; can be high if antibody or blocking is suboptimal | Non-specific binding to beads/GST can be significant without optimization | Can be controlled via crosslinking and stringent washing | Generally low background due to multiple affinity steps |
| Weak / transient interactions | Moderate; can be improved by buffer optimization or optional crosslinking | Often challenging; weak interactions frequently lost | Usually poor for very transient interactions | Good for crosslinked chromatin-bound complexes | Best for stable complexes; transient interactions may still be underrepresented |
| Throughput & scale | Medium; suitable for both individual targets and MS-based discovery | Medium; typically used for a limited number of targets | Low–medium; mostly used for targeted pairwise validation | Often used for large-scale chromatin-related interaction mapping | Used for in-depth analysis of a limited number of complexes |
| Typical readouts | WB or LC–MS/MS | Mainly WB; MS possible but background can limit depth | Mostly WB | LC–MS/MS | LC–MS/MS |
| Typical use cases | Tag-based capture of bait and interactors in cells; clean enrichment + WB/MS | Validation of endogenous interactions with available antibodies | In vitro binding/validation of defined protein pairs | Nuclear receptor/TF–chromatin interaction complexes | High-purity, stable complexes for deep proteomic characterization |
Project consultation & experimental design
Sample preparation / expression
Strep-Tactin affinity capture & washing
Mild elution & sample preparation for WB / MS
Readout: Western blot or LC–MS/MS
Data analysis, interpretation & reporting
Orbitrap LC–MS/MS for High-Confidence Interactome Profiling
Strep pull-down samples are analyzed on the Thermo Scientific Orbitrap Fusion Lumos™ Tribrid™ mass spectrometer coupled with nanoLC (EASY-nLC 1200), enabling sensitive, high-resolution profiling of complex affinity-enriched samples.
Together, this platform delivers high specificity, strong depth, and reproducible quantification, supporting publication-ready interaction datasets.
Controls & QC
| Item | Purpose | Pass criteria |
| Tag-only control | Remove tag/resin background | Minimal signal vs bait sample |
| Beads-only control | Remove matrix binders | Low non-specific carryover |
| Input → Elution tracking | Confirm enrichment & clean washes | Bait strong in elution; washes clean |
| Biological replicates | Statistical confidence | High overlap across replicates |
| MS QC (FDR) | Reliable IDs | Standard PSM/peptide/protein FDR applied |
Orbitrap Fusion Lumos Tribrid (Figure from Thermo)
| Sample type | Recommended for | What you provide | Notes |
| Cell lysate (preferred) | Most Strep pull-down projects (WB or MS) | Clarified lysate from cells expressing Strep-tagged bait | Best balance of yield and native interactions |
| Cell pellet | When you prefer us to perform lysis | Frozen cell pellets | Requires expression confirmation and handling notes |
| Tissue lysate / tissue | Tissue-specific interactomes | Clarified lysate or frozen tissue | May require additional optimization; higher complexity/background risk |
| Purified protein / recombinant bait | In vitro validation or controlled binding assays | Purified Strep-tagged bait (and optional prey) | Best for defined interactions, less "cell-context" |
| Item | Required details |
| Bait protein | Gene/protein name, species, expected MW, key domains (if relevant) |
| Tag details | Strep-tag type (Strep-tag II / Twin-Strep), N- or C-terminal, linker (if any) |
| Expression system | Cell line / organism, transient or stable expression, treatment conditions |
| Experimental groups | Control vs treatment; mutant vs WT; time points; number of replicates |
| Readout | WB, LC–MS/MS, or WB + MS |
| Controls | Tag-only/empty vector control (recommended), bead-only control (optional) |
| For WB projects | Target interactor list + antibody information (species/reactivity, catalog if available) |
Mechanistic and Signaling Pathway Studies
Map how receptors, adaptors and kinases assemble into signaling complexes to drive specific cellular responses.
Target Validation & Mechanism of Action (MoA)
Define the interaction partners of a drug target to understand how it functions and how interventions modulate its network.
Drug, Perturbation & Condition-Dependent Interactomes
Compare interaction profiles across treatments, knockdown/overexpression, stress conditions or time points to reveal dynamic changes.
Complex & Compartment-Specific Protein Networks
Characterize multi-protein complexes in defined cellular compartments (cytosol, nucleus, organelles) using optimized lysis and fractionation.
Domain, Mutant and Isoform Comparison
Assess how truncations, point mutations or alternative isoforms reshape the interactome of your bait protein.
Follow-up Validation of Screen or -Omics Hits
Use Strep pull-down–WB/MS to confirm candidate interactors identified by RIME-MS, AP-MS, proximity labeling or genetic/chemical screens.
Strep Pull-down + WB Projects
Strep Pull-down + LC–MS/MS Projects
Enrichment QC (Input/FT/Wash/Elution + tag-only)
Enrichment QC for Strep pull-down showing bait enrichment from input to elution with minimal signal in washes and tag-only control.
Interactor Validation
Western blot validation of candidate interactors co-enriched with the bait in elution fractions, with tag-only/beads-only controls confirming specificity.
MS Quality & Depth
LC–MS/MS quality overview showing overlapping TIC replicates and consistent identification depth across runs.
Volcano plot
Volcano plot (bait vs tag-only) highlighting significantly enriched interactors and the bait protein under defined statistical thresholds.
Why choose Twin-Strep-tag for LC–MS/MS discovery?
A. Twin-Strep-tag typically provides stronger capture than Strep-tag II, improving enrichment of low-abundance bait proteins and enabling more stringent washing for cleaner MS results.
Does N- vs C-terminal Strep-tag placement matter?
A. Yes. Tag position can affect folding or block binding interfaces, which may change the interactome. If unsure, test both orientations in a pilot WB pull-down.
How do you separate true interactors from background in Strep-MS?
A. We use negative controls (tag-only and/or beads-only), enrichment thresholds (e.g., log2FC > 1), replicate statistics, and optional SAINT scoring to prioritize high-confidence PPIs.
Can Strep pull-down be used for membrane protein interactomes?
A. Yes. With optimized detergent conditions, Strep-tag/Strep-Tactin capture can support membrane protein solubilization while maintaining robust enrichment for MS.
When should I use chemical crosslinking with Strep pull-down?
A. Crosslinking can help capture transient or weak interactions, but may increase background and analysis complexity. We recommend starting native and adding crosslinking only if needed.
Can Strep-Tactin pull down endogenous proteins without a tag?
A. Not typically. Strep pull-down is tag-dependent; for endogenous bait proteins, Co-IP with a specific antibody is usually required.
What is the minimum sample amount for Strep-MS?
A. A common starting point is ~10⁷ cells or 50–100 mg tissue per replicate, depending on bait abundance and study design.
Is the Strep-tag compatible with mass spectrometry?
A. Yes. The Strep-tag is small and generally does not interfere with peptide identification or database searching.
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