Protein G and Protein A Bind to Different IgG

The high affinity of protein G and protein A for the Fc region of polyclonal and monoclonal IgG-type antibodies forms the basis for purification of IgG, IgG fragments containing the Fc region, and IgG subclasses.

Protein G and protein A are bacterial proteins from Group G Streptococci and Staphylococcus aureus, respectively. When coupled to Sepharose, protein G and protein A create extremely useful, easy-to-use chromatography media for routine purification of antibodies. Examples include the purification of monoclonal IgG-type antibodies, purification of polyclonal IgG and its subclasses, adsorption and purification of immune complexes involving IgG, and fusion proteins. IgG subclasses can be isolated from cell culture supernatants, serum, and ascites.

Table 1 shows a comparison of the relative binding strengths of protein G and protein A to different immunoglobulins. The information has been compiled from various publications. Binding strengths are tested with free protein G or protein A and can be used as guidelines to predict the binding behavior to a protein G or protein A purification medium. However, when coupled to an affinity matrix, the interaction can be altered. For example, rat IgG1 binds to Protein G Sepharose, but not to Protein A Sepharose.

Single-step purification of samples from native sources or calf serum-supplemented medium based on Fc region specificity will co-purify host IgG and can even bind trace amounts of serum proteins. To avoid trace amounts of contaminating IgG, consider alternative techniques such as immunospecific affinity using anti-host IgG antibodies coupled to, for example, NHS-activated Sepharose, ion exchange chromatography (IEX) with, for example, Capto™ adhere or hydrophobic interaction chromatography.

Table 1. Relative binding strengths of antibodies from various species to protein G and protein A as measured in a competitive ELISA test. The amount of IgG required to give a 50% inhibition of binding of rabbit IgG conjugated with alkaline phosphatase was determined.

Species	Subclass	Protein G binding	Protein A binding
Human	IgA IgD IgE IgG1 IgG2 IgG3 IgG4 IgM*	– – – ++++ ++++ ++++ ++++ –	variable – – ++++ ++++ – ++++ variable
Avian egg yolk	IgY†	–	–
Cow		++++	++
Dog		+	++
Goat		++	–
Guinea pig	IgG1	++	++++
Hamster		++	+
Horse		++++	++
Koala		+	–
Llama		+	–
Monkey (rhesus)		++++	++++
Mouse	IgG1 IgG2a IgG2 IgG3 IgM*	++++ ++++ +++ +++ –	+ ++++ +++ ++ variable
Pig		+++	+++
Rabbit		+++	++++
Rat	IgG1 IgG2a IgG2 IgG3	+ ++++ ++ ++	– – – +
Sheep		++	+/–

* Purified using HiTrap IgM Purification HP columns.
^† Purified using HiTrap IgY Purification HP columns.
++++ = strong binding.
++ = medium binding.
— = weak or no binding.