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IP Preparation

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Binding Proteins & Beads

Protein A and Protein G are suited for detection of IP proteins as they work by binding to the intact Fc regions of IgGs. However, they are not interchangeable detection reagents; their use is determined by the isotype of the capture antibody as they exhibit differing isotype selectivity. Protein A/G binds to all subclasses of immunoglobulins.

There are two types of beads available:

– Agarose (Sepharose) beads

– Magnetic beads (commonly used)

 

Table 1
Affinity of Protein A and Protein G to different immunoglobin subclasses of human, mouse, rat, and guinea pig species.
Species Ig Subclass Protein A binding Protein G binding

Human

IgA

IgD

IgE

IgG1

IgG2

IgG3

IgG4

IgM

Variable

+

+

++++

++++

–

++++

Variable

–

–

–

++++

++++

++++

++++

–

Mouse

IgG1

IgG2a

IgG2b

IgG3

IgM

+

++++

+++

++

Variable

++++

++++

+++

+++

–

Rat

IgG1

IgG2a

IgG2b

IgG2c

IgG3

–

–

–

Variable

+

+

++++

++

++

++

Guinea Pig

 IgG

 ++++

 ++

++++       Strong Binding

+++/++   Medium Binding

Variable/– Weak or No Binding
 
Choosing The Right Primary Antibody

In IP, the target protein is usually in the native conformation. For this reason, antibodies used for IP need to recognize and have high affinity to the epitope on the surface of the protein.

What to consider before choosing the right antibody:

  • The immunogen is on the exposed surface of the protein (it can be checked if the structure of the protein and immunogen are known).

  • The antibody works for ChIP and/or IHC. These techniques, similar to IP, require antibodies that recognize surface epitopes in the native form.

  • Monoclonal antibodies recognize a single epitope and have minimal batch-to-batch variation.

  • Polyclonal antibodies recognize several epitopes and different affinities. Batch-to-batch variation is more likely compared to monoclonal antibodies.

Tip: Where possible, use a polyclonal antibody to capture the target protein. Polyclonals bind to multiple epitopes on the target protein, meaning a greater amount of protein is retained.

Antibody Pairs

The use of antibody pairs, i.e., a capture antibody from one species, and an antibody for WB detection from another, is an additional factor to consider when planning an IP experiment. The antibody selection process should ensure that both antibodies recognize different epitopes of the target protein, in addition to originating from different species. The antibody type (i.e., polyclonal or monoclonal) is important. The best option is a combination of a polyclonal capture antibody and a monoclonal antibody for detection. This combination ensures maximum capture efficiency with high detection specificity.