Protein Protocols Protein Protocols

390 Hong et al.
Table 1
Comparison of Staining Procedures for Protein Blots a
Approx. detection
Dyes Conditions limits (ng)
0.05% Coomassie S: 50% MeOH–7% HAc 100 d
Blue R-250 (10 min)
D: 10% MeOH–14% HAc
(overnight at 65°C)
0.1% Amido S: 45% MeOH–10% HAc 50 d
Black 10B (10 min)
D: 10% HAc (15 min)
0.1% Ponceau S S: 5% HAc (3 min) 100 d
W:Water b
0.05% CPTS S: 12 mM HCl (~1min) 10 d
W: 12 mM HCl b
0.008% DB71 S: 40% EtOH–10% HAc 5–10 (our work)
(5 min)
W: 40% EtOH–10% HAc c
a The staining procedures of Coomassie Blue (9), Amido Black (6), and CPTS (20)
were performed as in references cited. Ponceau S staining followed the product information
of Sigma Chemical.
b Membranes were briefly washed in several changes of the solutions to remove excess dye.
c Membranes were washed once in the solution, since background was almost clear.
d Approximate detection limits are from the references cited, where they were determined
by comparing protein slot blots stained by these methods.
S, staining; D, destaining; W, washing; MeOH, methanol; HAc, acetic acid; EtOH,
ethanol.
take up to an hour or more, depending on the amount or nature of proteins of interest. The
membranes can be repeatedly stained and destained, with no apparent loss of sensitivity.
10. Both Coomassie Blue and Amido Black are inferior to DB71 stain in terms of requiring
separate destaining solution and lengthy time (see Table 1), and giving lower sensitivity
on PVDF.
11. DB71 is comparable with copper phthaloryanine-3,4',4'',4'''-tetrasulfonic acid (CPTS) (see
Figs. 2C and G) reported as the highest sensitive dye-binding staining method (20), in its
sensitivity, rapidity, and reversibility. The bluish violet color of DB71 stained proteins
gives better band contrast than the turquoise blue CPTS stained ones, allowing easy photography
without any filter system; moreover, DB71 is much cheaper than CPTS.
12. It is noticeable that among the methods tested, DB71 staining allows the highest band
contrast on PVDF with a similar staining intensity as on NC (see Figs. 2D and H).
13. DB71 staining is applicable to a subsequent immunostaining without removing the dye
from developed bands. However, we recommend the dye removal, prior to the immunodetection
of small amounts of a particular protein.
14. DB71 staining can also be applied for quantification purposes with simplicity and convenience
over the conventional spectrophotometric procedures. The relationships between
peak area representing band intensity after DB71 staining and the protein amounts hold a
linearity between 20 and 1000 ng, with a correlation coefficient of 0.991 (see Fig. 3).
15. A higher band contrast for photography is obtained when the stained membrane keeps wet
rather than dry. The stain remains stable for several months in a refrigerator.