Affinity Tag Purification
High-throughput protein purification using engineered affinity tags for rapid isolation and concentration
Affinity tag purification is a cornerstone of our protein production pipeline, enabling rapid, high-purity isolation of target proteins from complex mixtures. Our platform supports multiple tag systems optimized for different experimental requirements.
Supported Affinity Tags
His-Tag
Polyhistidine tags for metal affinity chromatography
- 6x to 10x histidine repeats
- Ni²⁺, Co²⁺, or Cu²⁺ affinity resins
- Suitable for native and denaturing conditions
Strep-Tag
Strep-Tag II and Twin-Strep-Tag for biotin-based purification
- Mild elution conditions
- High specificity and low background
- Compatible with functional studies
FLAG-Tag
FLAG octapeptide for antibody-based purification
- Mild elution with FLAG peptide
- Excellent for protein complexes
- Minimal structural interference
GST-Tag
Glutathione S-transferase fusion for glutathione affinity
- Large tag aids in protein solubility
- Gentle elution conditions
- Suitable for protein-protein interactions
Tag Positioning Strategy
Our platform supports flexible tag placement to optimize protein function and purification efficiency.
Advantages:
- Easy genetic fusion
- Minimal disruption of protein domains
- Accessible for purification
- Compatible with most expression systems
Best for:
- Enzymes with C-terminal active sites
- Proteins with critical C-terminal domains
- Secreted proteins
- Membrane proteins (cytoplasmic tags)
Example applications:
- Binding domain studies
- Enzyme kinetics
- Structural characterization
Advantages:
- Easy genetic fusion
- Minimal disruption of protein domains
- Accessible for purification
- Compatible with most expression systems
Best for:
- Enzymes with C-terminal active sites
- Proteins with critical C-terminal domains
- Secreted proteins
- Membrane proteins (cytoplasmic tags)
Example applications:
- Binding domain studies
- Enzyme kinetics
- Structural characterization
Advantages:
- Preserves native N-terminus
- Ideal for proteins with N-terminal signal sequences
- Better for some protein classes
- Alternative when N-terminal tags interfere
Best for:
- Proteins with N-terminal functional domains
- Signal peptide-containing proteins
- Proteins requiring native N-terminus
- Secreted protein studies
Example applications:
- Membrane protein studies
- Signal peptide analysis
- Native protein behavior studies
Advantages:
- Minimal functional disruption
- Preserves native termini
- Can be placed in flexible loops
- Useful for difficult proteins
Best for:
- Proteins where terminal tags interfere
- Structural studies requiring native ends
- Functional studies
- Proteins with critical terminal domains
Considerations:
- Requires structural knowledge
- May need loop engineering
- More complex design process
Purification Workflow
Protein Expression
Proteins are expressed with chosen affinity tags using cell-free or cellular systems.
Tag selection is optimized based on downstream applications and protein characteristics.
Cell Lysis and Clarification
Cells are lysed under appropriate conditions to preserve protein integrity and tag accessibility.
Lysis conditions are optimized for each tag system to maximize binding capacity.
Affinity Capture
Clarified lysate is applied to affinity resin under optimized binding conditions.
Binding conditions:
- Buffer composition optimized for each tag
- pH and salt concentration controlled
- Temperature maintained for stability
- Binding time optimized for capacity
Washing
Extensive washing removes non-specific binding proteins and contaminants.
Elution
Target proteins are eluted using tag-specific conditions.
- Imidazole gradient: 10-500 mM imidazole
- pH elution: Low pH (4.0-5.0)
- Competitive elution: High histidine concentration
- Imidazole gradient: 10-500 mM imidazole
- pH elution: Low pH (4.0-5.0)
- Competitive elution: High histidine concentration
- Desthiobiotin: 2.5-10 mM in elution buffer
- Biotin: 1-5 mM (stronger elution)
- Gentle conditions: Maintains protein activity
- FLAG peptide: 100-500 μg/mL
- Glycine-HCl: pH 3.0-3.5
- Competitive elution: Preserves structure
Typical purification yields range from 1-50 mg per liter of culture, depending on protein and expression system.
Quality Assessment
Purity Analysis
- SDS-PAGE gel analysis
- Protein concentration determination
- Contaminant identification
- Yield calculations
Functional Assessment
- Activity measurements
- Binding capacity tests
- Structural integrity checks
- Stability evaluations
Tag Removal
- Protease cleavage sites
- Enzymatic tag removal
- Purification of cleaved protein
- Verification of removal
Batch Consistency
- Reproducibility testing
- Batch-to-batch comparison
- Quality control metrics
- Process optimization
Advanced Purification Strategies
Multi-Tag Systems
For challenging proteins, we employ multiple complementary tags:
- Dual affinity tags: His-FLAG or His-Strep combinations
- Sequential purification: Stepwise purification for high purity
- Orthogonal tags: Different chemical principles for specificity
Specialized Applications
Integration with Downstream Analysis
Purified proteins are immediately compatible with:
- Binding assays: Direct use in BLI and SPR studies
- Structural analysis: Clean samples for crystallography
- Functional studies: Activity measurements and characterization
- Quantification: Accurate concentration determination
Tag removal may be necessary for some applications. Consider the impact of tags on protein function when designing experiments.
Our team provides guidance on tag selection, positioning, and purification optimization based on your specific protein and experimental requirements.