Protein-A/G/L-Cys
Description
Product Specs
Q&A
Structural Composition and IgG Binding Specificity
Q. What structural elements determine the IgG binding specificity of Protein-A/G/L-Cys?
The protein comprises three modular domains:
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5 IgG-binding domains from Protein A (targeting Fc regions of human, rabbit, and pig IgG)
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2 IgG-binding domains from Protein G (extending binding to cow, goat, and sheep IgG)
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5 κ light chain-binding domains from Protein L (enabling interactions with Fab regions of IgA, IgM, and scFv fragments)
Methodological Validation:
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Surface Plasmon Resonance (SPR) quantifies binding kinetics across species (e.g., human IgG1: K<sub>D</sub> = 10<sup>−9</sup> M; mouse IgG2a: K<sub>D</sub> = 10<sup>−8</sup> M) .
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ELISA with Subtype Panels: Pre-block plates with 0.1% BSA to minimize non-specific adsorption, then assess binding to IgG subtypes (Table 1).
Table 1. IgG Binding Affinity by Species
Reconstitution and Stability Protocols
Q. How should researchers handle Protein-A/G/L-Cys to maintain functionality?
Reconstitution:
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Dissolve lyophilized powder in distilled water (pH 7.0–7.4) to 1–5 mg/mL.
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Avoid vortexing; use gentle pipetting to prevent aggregation .
Storage:
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Short-term: 4°C (2–7 days) with 0.1% BSA to prevent adsorption .
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Long-term: Aliquot at ≤−20°C; limit freeze-thaw cycles to ≤3 .
Stability Metrics:
Immunoprecipitation Optimization for Low-Abundance Antibodies
Q. How can binding efficiency be enhanced for rare antibodies in complex matrices?
Carrier Protein Strategy:
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Pre-incubate magnetic beads with 1% BSA for 1 hr to block non-specific sites.
Buffer Optimization:
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Binding Buffer: 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 (pH 8.0).
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Elution: Compare 0.1 M glycine (pH 2.5) vs. 0.5 M arginine (pH 6.5) to preserve antibody integrity.
Data Contradiction Analysis:
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If recovery varies between serum and cell culture supernatants:
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Treat DNA-rich samples with DNase I (1 U/μL, 37°C, 30 min).
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Normalize lipid content via ultracentrifugation (100,000 × g, 1 hr).
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Resolving Species-Specific Binding Discrepancies
Q. Why does bovine IgG show weak binding despite Protein G domains?
pH Adjustment: Lower buffer pH to 5.0 to enhance Fc-Protein G interactions.
Validation Workflow:
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Western Blot: Use anti-κ light chain antibodies to confirm target presence.
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Competitive ELISAs: Titrate free κ light chains (0–100 μM) to quantify binding occupancy .
Table 2. Binding Efficiency Under Adjusted pH
| Species | pH 7.0 Binding (%) | pH 5.0 Binding (%) |
|---|---|---|
| Bovine | 15 ± 3 | 82 ± 7 |
| Human | 98 ± 2 | 95 ± 3 |
| Data from |
Cysteine-Driven Conjugation for Affinity Chromatography
Q. How does the C-terminal cysteine enable site-directed immobilization?
Coupling Chemistry:
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Reduce disulfide bonds with 2 mM TCEP (30 min, 25°C).
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Immobilize on maleimide-activated agarose (2–10 mg protein/mL resin).
Performance Metrics:
Recommendations:
Product Science Overview
Protein A/G/L-Cys Recombinant is composed of:
- Five IgG-binding regions of Protein A: Derived from Staphylococcus aureus segments E, D, A, B, and C.
- Two IgG-binding regions of Protein G: Derived from Streptococcus segments C1 and C3.
- Five IgG-binding regions of Protein L: Derived from Peptostreptococcus magnus segments B1, B2, B3, B4, and B5 .
The recombinant protein is a single non-glycosylated polypeptide chain containing 806 amino acids and has a molecular mass of approximately 89.3 kDa . It is fused with a cysteine (Cys) residue at the C-terminus to facilitate conjugation and immobilization .
The recombinant protein has been engineered to eliminate non-specific binding regions, such as cell wall binding, cell membrane binding, and albumin binding regions . This ensures maximum specific IgG binding. Protein A/G/L-Cys Recombinant can bind to IgG antibodies from a wide range of species, including human, mouse, rat, cow, goat, sheep, rabbit, guinea pig, pig, dog, and cat .
Protein A/G/L-Cys Recombinant is widely used in various applications, including:
The lyophilized protein is stable at room temperature for up to three weeks but should be stored desiccated below -18°C for long-term storage . Upon reconstitution, it should be stored at 4°C for short-term use (2-7 days) and below -18°C for long-term use. To prevent freeze-thaw cycles, it is recommended to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) .
