Protein Protocols Protein Protocols

1100 Johansen and Svensson
Table 3
Specificity of Protein A, Protein G, and Jacalin
for Immunoglobulins from Various Species a,b
Antibodies Protein A Protein G type 2 Jacalin
Human IgG 1 ++ ++ –
Human IgG 2 ++ ++ –
Human IgG 3 – ++ –
Human IgG 4 ++ ++ –
Human IgA 1 + – ++
Human IgA 2 + – ?
Rabbit ++ ++
Cow + ++
Horse – ++
Goat + ++
Guinea pig ++ ++
Sheep – ++
Dog ++ ++
Pig ++ ++
Mouse IgG + ++
Mouse IgA ? ? –
Rat – +
a Reproduced with the permission of Pharmacia Biotech from the data file
for GammaBind G, type 2.
b Binding capacity: ++, strong; +, intermediate; –, weak.
1.3. Formation of Antigen–Antibody Complexes
1.3.1. Direct Immunoprecipitation
The antibody or antigen preparation to be tested by immunoprecipitation is now
allowed to react with aliquots of the cell lysate or a specific antibody. The antibody–
antigen complex is then precipitated by one of several methods. Depending on
the antibody to be precipitated, staphylococcal protein A.
Protein A consists of a single polypeptide chain with two accessible high-affinity binding
sites for the Fc region of IgG and secondary binding sites for the Fab region. The
protein A antibody complexes disassociate at low pH, boiling, and/or the addition of
reducing reagents. Protein A binds a relatively broad spectrum of antibodies and antibody
subclasses from different species (Table 3). One milliliter of protein A-Sepharose
beads can adsorb 10–20 mg of IgG. The specificity of antibody capture applications can be
manipulated by adjusting the pH and ionic strength of the binding and elution buffers (7–9).
Native and recombinantly engineered protein G bind specifically to more species of
immunoglobulins than does protein A (Table 3). The recombinant form does not
crossreact with IgM, IgE, IgA, IgD, or serum albumin as protein A may do (10–12).
Jacalin, purified from the jack-fruit Artrocarpus integrifolia, bound to Agarose beads,
has recently been introduced for the purification of human monomeric immunoglobulin
A, subclass 1 (Table 3) (13,14). Jacalin is a lectin composed of four identical subunits
of approx 10,000 Da each. This glycoprotein appears to bind only O-glycosidically linked