optimized and consistently manufactured to maximize reproducibility. Always bear in mind that the sensitivity of stains may
be dependent on each protein’s chemical composition, and
practical detection limits for some proteins may be 10Â higher
than the optimal case.
The most sensitive colorimetric method is silver staining
(optimally ~0.5 ng/band) [49, 50]. Proteins are usually fixed
in the gel using trichloroacetic acid (TCA) and then incubated
in a staining solution of silver nitrate. Silver ions bind to carboxylic acid, imidazole, and sulfhydryl sidechain groups and to
amines. The subsequent development process reduces the
protein-bound silver ions quickly to metallic silver, resulting
in brown-black colored bands (although formulations exist to
vary the color depending on protein charge and other features). A pH change is used to stop the reaction before all silver
ions in the gel are reduced. The specificity and efficiency of
silver ions binding to proteins as well as effective development
require various sensitizers and enhancers, some of which (e.g.,
glutaraldehyde, formaldehyde) chemically crosslink the proteins in the gel matrix, limiting destaining and elution for
downstream MS.
Reverse zinc staining is also highly sensitive (~1 ng/band)
and works by staining areas not containing SDS-bound protein
[51]; thus, bands are clear in an opaque background. Zinc ions
bind to imidazole buffer, and the resulting complex precipitates in the gel matrix except where SDS-saturated proteins are
located. Staining is fast (15 min), no fixation steps are needed,
and the stain can be easily removed.
Fluorescent dye stains can be used if a fluorescence imaging
system is available. The stains have high sensitivity (in the range
3–10 ng/band dependent on their chemistry) and high reproducibility. Most fluorescent stains are based on a noncovalent
dye-binding mechanism, allowing complete destaining and
recovering of the protein. SYPRO family dyes, which exhibit
fluorogenic enhancement when interacting with hydrophobic
groups of protein or SDS bound to protein, are commonly
used, but alternative families of molecules such as LUCY,
Krypton, and Flamingo may offer advantages of photostability
and sensitivity.
Glycoproteins or phosphoproteins carry certain chemical
moieties to which functional group-specific stains can be coupled. Other stains are able to selectively stain His-tags in PA
gels without the need for His-tag-specific antibodies. However,
such stains are not typically used in a QC context as they will
only reveal the targeted subset of proteins and not
contaminants.
In commercial, stain-free methods, the intrinsic fluorescence of Trp residues is enhanced by UV-light-controlled
42
Bertrand Raynal et al.
be dependent on each protein’s chemical composition, and
practical detection limits for some proteins may be 10Â higher
than the optimal case.
The most sensitive colorimetric method is silver staining
(optimally ~0.5 ng/band) [49, 50]. Proteins are usually fixed
in the gel using trichloroacetic acid (TCA) and then incubated
in a staining solution of silver nitrate. Silver ions bind to carboxylic acid, imidazole, and sulfhydryl sidechain groups and to
amines. The subsequent development process reduces the
protein-bound silver ions quickly to metallic silver, resulting
in brown-black colored bands (although formulations exist to
vary the color depending on protein charge and other features). A pH change is used to stop the reaction before all silver
ions in the gel are reduced. The specificity and efficiency of
silver ions binding to proteins as well as effective development
require various sensitizers and enhancers, some of which (e.g.,
glutaraldehyde, formaldehyde) chemically crosslink the proteins in the gel matrix, limiting destaining and elution for
downstream MS.
Reverse zinc staining is also highly sensitive (~1 ng/band)
and works by staining areas not containing SDS-bound protein
[51]; thus, bands are clear in an opaque background. Zinc ions
bind to imidazole buffer, and the resulting complex precipitates in the gel matrix except where SDS-saturated proteins are
located. Staining is fast (15 min), no fixation steps are needed,
and the stain can be easily removed.
Fluorescent dye stains can be used if a fluorescence imaging
system is available. The stains have high sensitivity (in the range
3–10 ng/band dependent on their chemistry) and high reproducibility. Most fluorescent stains are based on a noncovalent
dye-binding mechanism, allowing complete destaining and
recovering of the protein. SYPRO family dyes, which exhibit
fluorogenic enhancement when interacting with hydrophobic
groups of protein or SDS bound to protein, are commonly
used, but alternative families of molecules such as LUCY,
Krypton, and Flamingo may offer advantages of photostability
and sensitivity.
Glycoproteins or phosphoproteins carry certain chemical
moieties to which functional group-specific stains can be coupled. Other stains are able to selectively stain His-tags in PA
gels without the need for His-tag-specific antibodies. However,
such stains are not typically used in a QC context as they will
only reveal the targeted subset of proteins and not
contaminants.
In commercial, stain-free methods, the intrinsic fluorescence of Trp residues is enhanced by UV-light-controlled
42
Bertrand Raynal et al.