Protein-A/G, His
Description
Binding Specificity and Applications
Protein-A/G, His exhibits broad cross-species IgG binding, making it ideal for:
-
Purification of monoclonal and polyclonal IgG antibodies from human, mouse, rabbit, rat, cow, goat, sheep, horse, and dog sera .
-
Detection of antibodies in ELISA, Western blotting, and immunoprecipitation .
-
Immobilization of antibodies for affinity chromatography or biosensor applications .
Table 1: Binding Specificity of Protein-A/G, His vs. Protein A and Protein G
| Species | IgG Subclass Bound | Protein A | Protein G | Protein-A/G, His |
|---|---|---|---|---|
| Human | IgG1, IgG2, IgG3, IgG4 | + | +++ | +++ |
| Mouse | IgG1, IgG2a, IgG2b, IgG3 | ++ | +++ | +++ |
| Rabbit | Total IgG | +++ | +++ | +++ |
| Cow | Total IgG | - | +++ | +++ |
| Goat | Total IgG | + | +++ | +++ |
Note: "+" indicates moderate binding, "+++" indicates strong binding. Protein-A/G, His binds all subclasses of human and mouse IgG, unlike Protein A (which weakly binds IgG3) or Protein G (which binds IgG3 strongly) .
Table 2: Key Differences Between Protein A, Protein G, and Protein-A/G, His
| Feature | Protein A | Protein G | Protein-A/G, His |
|---|---|---|---|
| Native Source | Staphylococcus | Streptococcus | Recombinant |
| Molecular Weight | 46,700 Da | 21,600 Da | 50,460–56,900 Da |
| Binding Regions | 5 Fc domains | 2 Fc domains | 4 + 2 Fc domains |
| Albumin Binding | No | No | No |
| Optimal pH | 8.2 | 5 | 5–8.2 |
Protein-A/G, His combines the advantages of both parent proteins:
-
Broader IgG subclass coverage (e.g., human IgG3 and mouse IgG2a) .
-
Reduced pH dependency compared to Protein A, enabling binding under acidic conditions .
-
Enhanced stability due to the absence of cell wall/membrane-binding regions .
Table 3: Production and Quality Metrics
| Parameter | Specification | Source |
|---|---|---|
| Host Organism | E. coli | |
| Purity | >96% (SDS-PAGE/HPLC) | |
| Storage Conditions | -18°C (lyophilized) | |
| Reconstitution Buffer | Sterile H₂O or neutral pH |
The His tag facilitates high-efficiency purification using nickel or cobalt affinity columns, achieving >95% purity in a single step .
Aggregation Inhibition
Protein A (but not Protein G) binds to a hydrophobic site in IgG’s Fc region, inhibiting agitation-induced aggregation. Protein-A/G, His may leverage this property to stabilize therapeutic antibodies during formulation .
Immunodiagnostic Applications
A Protein-A/G, His-based ELISA demonstrated high sensitivity and specificity for detecting Pythium insidiosum antibodies in human, horse, dog, and cow sera, validating its utility in cross-species immunodiagnosis .
Comparative Binding Affinity
Protein G exhibits stronger binding to human IgG3 and rat IgG2a than Protein A, but Protein-A/G, His retains this specificity while expanding its range .
Product Specs
(a) Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) analysis.
(b) Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis.
Product Science Overview
Protein A/G is composed of five IgG-binding regions from Protein A (E, D, A, B, and C) and three from Protein G (C1, C2, and C3). This combination results in a protein with a total of 513 amino acids and a molecular mass of approximately 56.9 kDa . The recombinant protein is produced in Escherichia coli and is fused with a 6×His tag at the C-terminus, which facilitates its purification and detection .
The recombinant Protein A/G has a broader binding capacity compared to either Protein A or Protein G alone. It can bind to various subclasses of human, mouse, and rat IgGs, including human IgG1, IgG2, IgG3, and IgG4; mouse IgG2a, IgG2b, and IgG3; and rat IgG2a and IgG2c . Additionally, it can bind to total IgG from other species such as cow, goat, sheep, horse, rabbit, guinea pig, pig, dog, and cat .
Protein A/G Recombinant, His Tag is widely used for the purification of monoclonal and polyclonal IgG antibodies. The presence of the His tag allows for easy purification using immobilized metal affinity chromatography (IMAC), which exploits the affinity of histidine residues for metal ions like nickel, cobalt, and copper . This method provides a highly efficient means of isolating the recombinant protein from complex mixtures.
The production of His-tagged proteins involves inserting a DNA sequence encoding a string of six to nine histidine residues into the vector used for protein expression. This results in the expression of a recombinant protein with a 6xHis or poly-His tag fused to its N- or C-terminus . The His tag facilitates the purification process, allowing for the specific binding and elution of the protein using metal-chelate supports .
Lyophilized Protein A/G 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 degradation, it is recommended to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) and to avoid repeated freeze-thaw cycles .
