Introduction
Choosing between Protein A vs Protein G for antibody quantification affects the reliability of the result. Both are bacterial Fc-binding proteins that recognise immunoglobulins, but they differ in which species and IgG subclasses they bind, and how strongly.
Understanding these differences matters. A capture molecule that binds the target antibody only weakly can underestimate concentration or produce inconsistent results between experiments. This guide explains the key differences and provides practical guidance on which to choose for different workflows.
What are Protein A and Protein G?
Protein A is a surface protein originally derived from Staphylococcus aureus. It binds to the Fc region of IgG antibodies, primarily at the CH2–CH3 interface. It has five Fc-binding domains and shows strong affinity for human IgG1, IgG2 and IgG4.
Protein G is a surface protein from Streptococcus species (Group C and G). It also binds the Fc region of IgG but at a slightly different site, and offers broader species reactivity, particularly for mouse antibodies where Protein A has notable gaps.
Both proteins are widely used in purification as affinity chromatography ligands, and in quantification as capture molecules on assay surfaces, sensors and plates. The key practical difference lies in their binding profiles.
Protein A vs Protein G: species and subclass binding
The table below summarises typical binding strength for the most commonly quantified antibody types. Binding is rated as strong (+++), moderate (++), weak (+) or negligible (–). Values are based on published data; confirmation against internal references is recommended before publishing.
| Antibody | Protein A | Protein G |
| Human IgG1 | +++ | +++ |
| Human IgG2 | +++ | +++ |
| Human IgG3 | – | +++ |
| Human IgG4 | +++ | +++ |
| Mouse IgG1 | + | +++ |
| Mouse IgG2a | +++ | +++ |
| Mouse IgG2b | ++ | ++ |
| Mouse IgG3 | + | ++ |
| Rat IgG1 | – | + |
| Rat IgG2a | – | ++ |
| Rabbit IgG | +++ | ++ |
| Goat IgG | – | ++ |
| Sheep IgG | – | ++ |
Protein A is the stronger binder for human IgG1, IgG2 and IgG4, and for rabbit IgG. Protein G is the broader option, particularly useful for mouse IgG1, human IgG3 and antibodies from goat, sheep or rat.
When to use Protein A for antibody quantification
Protein A is a common choice when:
- The antibody is human IgG1, IgG2 or IgG4, the most common isotypes in therapeutic antibody development
- The workflow involves rabbit polyclonal or monoclonal antibodies
- The focus is on human antibody quantification and broad species coverage is not required
Protein A is the standard capture molecule in most human antibody manufacturing and purification workflows. Using it for quantification keeps the measurement aligned with the process chemistry.
When to use Protein G for antibody quantification
Protein G is a common choice when:
- The antibody is mouse IgG1, the most common isotype produced by murine hybridomas and a notable weak spot for Protein A
- The sample contains mixed species or the species is uncertain
- The workflow involves mouse-derived antibodies from hybridoma screening or cell line development
- The antibody is human IgG3, which Protein A binds poorly
- The antibody is from goat, sheep or rat
Protein G’s broader reactivity makes it a useful default when sample composition is variable. In panel screening workflows where isotype distribution may not yet be known, Protein G tends to give more consistent capture across the panel.
Practical considerations
Can both be used in the same experiment? Parallel measurement with Protein A and Protein G can be informative during method development, particularly to confirm that the capture molecule is not the limiting factor in assay sensitivity. Significant differences between the two can indicate that the antibody’s Fc region has a binding preference that should be accounted for.
Does binding strength affect accuracy? Yes. Weak or partial binding at the capture surface can underestimate the true antibody concentration. Choosing the right capture molecule for the specific antibody directly affects the reported titre values.
What about Fc-engineered antibodies? Antibodies with modified Fc regions, such as knobs-into-holes bispecifics, aglycosylated variants or Fc-silenced constructs, may have altered binding to both Protein A and Protein G. Empirical testing is recommended to confirm capture efficiency before relying on either.
What about IgM, IgA or non-IgG immunoglobulins? Neither Protein A nor Protein G binds IgM, IgA, IgD or IgE with meaningful affinity. For non-IgG immunoglobulin quantification, an anti-isotype antibody in an ELISA or similar immunoassay format is typically used.
Protein A and Protein G on Amperia™
Abselion offers Amperia™ sensor strips coated with either Protein A or Protein G, allowing the capture chemistry to be matched to the antibody:
- Protein A sensors, validated for human IgG quantification
- Protein G sensors, validated for human and mouse IgG quantification
Both sensor types integrate into the existing Amperia™ workflow without changes to sample preparation or data analysis. The system’s software applies the appropriate calibration model for the selected sensor type.
Which to choose depends on the specific antibody. Protein G’s broader species reactivity can make it a useful option when working with uncertain or mixed samples.
Summary
| Protein A | Protein G | |
| Best for | Human IgG1/2/4, rabbit IgG | Mouse IgG1, human IgG3, broad species coverage |
| Weakest for | Mouse IgG1, human IgG3, goat/sheep/rat | Slightly weaker than Protein A for human IgG1/2/4 |
| Often chosen when | Working with human therapeutics | Species or isotype is uncertain |
Frequently asked questions
Is Protein A or Protein G better for mouse antibodies?
Protein G is generally the better choice for mouse antibodies, particularly mouse IgG1, which binds only weakly to Protein A. For mouse IgG2a, both are effective. When the isotype is unknown or mixed, Protein G offers more consistent capture.
Can Protein A be used for rat IgG?
Protein A binds rat IgG only weakly or not at all, depending on the subclass. For rat antibody quantification, Protein G is typically the more reliable choice.
What is the difference between Protein A and Protein G binding sites?
Both bind the Fc region of IgG, but at slightly different positions. Protein A primarily binds at the CH2–CH3 interface of the Fc region, while Protein G binds at a related but distinct site. This difference in binding location contributes to their different specificities across species and subclasses.
Do Protein A and Protein G bind IgM or IgA?
Neither binds IgM, IgA, IgD or IgE with meaningful affinity. They are specific to IgG. For non-IgG immunoglobulins, an anti-isotype antibody in an ELISA format is typically used instead.
Which should I use if I don’t know the antibody isotype?
Protein G is usually the safer starting point due to its broader species and isotype reactivity. Once the isotype is confirmed, the method can be validated with the more specific capture molecule if needed.