Protein Protocols Protein Protocols

410 Kruger
Brilliant Blue is unsuitable for nitrocellulose membranes, since generally it produces
heavy background staining (see Note 14).
3.3.1. Amido Black
Incubate the filter for 2–5 s in 100 mL of stain solution. Transfer immediately to
100 mL of destain solution, and wash with several changes to remove excess dye.
Unacceptably dark backgrounds are produced by longer incubation times in the stain
solution.
3.3.2. Ponceau S
Incubate the filter with 100 mL of Ponceau S stain solution for 30 min. Wash excess
dye off the filter by rinsing in several changes of distilled water. The proteins may be
destained by washing the filter in PBS (see Note 7).
4. Notes
1. Protein G, a cell wall component of group C and group G streptococci, binds to the Fc
region of IgG from a wider range of species than that recognized by protein A (8). Therefore
antibodies that react poorly with protein A, particularly those from rat, mouse, goat,
and sheep, may be detected by a similar method using protein G derivatives. Natural protein
G also contains albumin binding sites and membrane binding regions which can lead
to nonspecific staining. However, these problems can be avoided by using protein G', a
recombinant, truncated form of the enzyme which lacks both albumin and membrane binding
sites and is thus more specific for IgG than the native form of the protein.
Protein L from Peptostreptococcus magus has affinity for κ light chains from various
species, and will bind to IgG, IgA, and IgM as well as Fab, F(ab') 2 and recombinant scFv
fragments that contain κ light chains. It will also bind chicken IgG. However, species such
as cow, goat, sheep, and horse whose immunoglobulins contain predominantly λ chains
will not bind well, if at all, to protein L (9). This problem has led to the development of
protein LA, a recombinant fusion protein that combines Fc- and Fab-binding regions of
protein A with κ light chain binding regions of protein L. This generates a molecule that
combines the favorable binding properties of both proteins (10).
Currently, alkaline phosphatase and horseradish peroxidase conjugated and 125I-labeled protein G are commercially available, while only peroxidase conjugates of protein L and
protein LA are produced.
2. Several visualization systems have been developed for both alkaline phosphatase and
horseradish peroxidase. Other colorimetric assay systems are described by Tijssen (4).
However, currently, greatest sensitivity is provided by chemiluminescent detection systems
(see Chapter 56). The alkaline phosphatase system is based on the light emission that
occurs during the hydrolysis of AMPPD (3-[2'-spiroadamantane]-4-methoxy-4-[3"phosphoryloxy]-phenyl-1,2-dioxetane).
The mechanism involves the enzyme catalyzed
formation of the dioxetane anion, followed by fragmentation of the anion to
adamantone and the excited state of methyl m-oxybenzoate. This latter anion is
the source of light emission. The peroxidase detection system relies on oxidation
of luminol (3-aminophthalhydrazine) by hydrogen peroxide, or other suitable
substrates, to produce a luminol radical. This radical subsequently forms an endoperoxide
that on decomposition, generates an electronically excited 3-aminophthalate dianion that
emits light on decay to its ground state. Light emission from this system can be enhanced
by the presence of 6-hydroxybenothiazole derivatives or substituted phenols, which act as
electron-transfer mediators between peroxidase and luminol.