Biomedical Engineering Reference
In-Depth Information
2.4 Staining Protocols
2.4.1 Staining with Organic Dyes
Because the ability to dye one protein may differ from that of
another in an electropherogram, an unknown protein mix should
become stained by different methods. Some of the procedures can
be done sequential in the same gel, e.g., additional Coomassie
staining after silver staining.
A synopsis of dyes used for visualization after electrophoresis
is given in Table 2.14.
Staining can also be used for quantifying proteins in gels. The
gel is scanned and the obtained picture is calculated by densito-
Densitometry
metric software. The amount of protein is proportional to the peak
area of the whole band. Since the reaction of a protein with a dye
can vary in a broad range (row “Bradford” in Table 1.1 corresponds
to Coomassie staining), calibration of the intensity is only possible
when the same or a very similar protein is used.
Qualitative interpretation of stained gels is often misleading,
since a blank area has not to be protein-free and an intensive band
has not to reflect a large amount.
Table 2.14.
Selected dyes suitable for electrophoresis
Dye
Staining of
Color
Proteins
Nucleic acids
index
Acridine Orange
x (RNA)
46005
Amido Black 10B
x
20470
8-Anilino-1-naphthal-
x
enesulfonic acid (ANS)
Bismarck Brown R
x (together
21010
with Coomassie)
Coomassie Blue G250
x
42655
Coomassie Blue R250
x (more sensitive
42660
and R350
than G250)
Coomassie Violett
x (for IEF)
42650
Ethidium bromide
x (fluorescence)
Fast Green FCF
x
42053
Methylen Blue
x (RNA)
52015
Methyl Green
x (native DNA)
42590
Ponceau S
x (Western
27195
blots on NC)
Stains All
x
x
Sudan Black
x (for lipoproteins)
26150
Sulforhodanin B
x (Western blots
45100
on PVDF)
