How do you choose protein A or G?

Protein A and G are structurally very similar, but they have slightly different affinities for IgG subclasses across different species. These affinities overlap, but in general, protein A has greater affinity for rabbit, pig, dog, and cat IgG whereas protein G has greater affinity for mouse and human IgG.

What does Protein G bind?

Protein A and protein G are bacterial proteins that bind human IgG, but also IgG from various other species. The proteins are widely used as affinity matrices for purification of IgG.

How does Protein G work?

Protein A/G is a recombinant fusion protein that combines IgG binding domains of both Protein A and Protein G. In addition, it binds to IgA, IgE, IgM and (to a lesser extent) IgD. Protein A/G also binds to all subclasses of mouse IgG but does not bind mouse IgA, IgM or serum albumin.

How do you elute from Protein G?


  1. Wash the Protein A or Protein G resin with at least 10 column volumes of 0.1 m TBS or 1× PBS.
  2. Dilute the serum 1:1 with the buffer used to wash the column.
  3. Pass the serum over the column at least two times using either gravity or a pump.
  4. Wash the column extensively until no protein can be detected in the eluent.

Does Protein G bind to IgM?

IgM does not bind to common bacterial protein A and protein G1, which are often used in co-immunoprecipitation applications.

What is Protein A column?

Protein A is a cell-wall protein of Staphylococcus aureus that can bind immunoglobulins from many species. Protein A columns are used for the purification of antibodies from complex mixtures such as serum, ascites, and hybridoma culture media. Several protein A column and cartridge formats are available.

Why does one use protein A or G in immunoprecipitation?

Protein A/G (or Protein A or Protein G) binds to the Fc region of an IP antibody to immobilize it in the correct orientation to immunoprecipitate the target antigen. Specificity of antibody-binding proteins. Proteins used to immobilize antibodies to beaded support show specificity to different antibody domains.

What is protein A column?

What is protein G column?

Protein G Sepharose® Columns are prepared by covalently coupling recombinant Protein G to 6% cross-linked Sepharose® beads. Protein G is a genetically engineered protein containing three IgG-binding regions of native Protein G. This product can be used for IgG purification and immunoprecipitation.

What is elution buffer used for?

Elution buffer is a major solvent in affinity chromatography. Elution buffer is used to wash away unbound proteins at first and at a greater concentration it releases the desired protein from the ligand.

What are protein A resins?

GenScript Protein A Resin is an affinity chromatography medium designed for easy, one-step purification of classes, subclasses and fragments of immunoglobulins from biological fluids and from cell culture media. Protein A Resin can also be used for immunoprecipitation of proteins,protein complexes or antigens.

How does protein A column work?

Protein A Chromatography relies on the specific and reversible binding of antibodies to an immobilized protein A ligand. Protein A is a 56 kDa surface protein of Staphylococcus aureus. Protein A resins are the most frequently used affinity resins in biomanufacturing.

What is the binding capacity of Dynabeads protein G?

Dynabeads® Protein G have a binding capacity of approximately 8 μg human IgG per mg beads. The amount of Ab captured depends on the concentration of Ab and Dynabeads® Protein G in the starting sample. Table 1. Binding strength of Protein A and G to different species of Igs and their subclasses

Which is the maximum binding capacity of protein?

SBC is usually reported as the maximum amount of protein bound to a chromatography resin at given solvent and protein concentration conditions. In these experiments, an excess of protein is loaded to give a maximum protein binding capacity.

How to determine dynamic binding capacity of chromatography?

In protein purification, dynamic binding capacity (DBC) of a chromatography column describes the maximum amount of target protein that you can load onto your column without causing unnecessary loss, measured under realistic experimental conditions (default flow-rate, real protein sample). Fig 1.

How does the SBC relate to protein binding capacity?

In contrast, although the SBC reports the maximum protein quantity that a resin can bind, it does not state the amount of protein that failed to bind under these conditions. Therefore, you may experience substantial losses of protein to the flow-through when using SBC as a guide for loading your protein.