ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 338 (2005) 159–161
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Notes & Tips
High-eYciency staining of proteins on diVerent blot membranes
C.R. Yonan, P.T. Duong, F.N. Chang¤
Department of Biology, Temple University, Philadelphia, PA 19122, USA
Received 18 June 2004
Available online 7 December 2004
Immunodetection of proteins on blot membranes, Wrst
developed by Towbin et al. [1], is the method of choice for
identifying speciWc proteins after electrophoresis. Due to
its high resolution, immunoprobing of blots from twodimensional polyacrylamide gels has been used extensively as a means of localizing speciWc proteins among
hundred or even thousands of separated proteins [2].
Three major types of membranes are used for protein
blotting: nitrocellulose, nylon, and polyvinylidene diXuoride (PVDF).1 Typically, before immunodetection, a blot
is subjected to a general protein stain such as Amido black
10B [3], Ponceau S [3], Coomassie brilliant blue R-250 [4],
India ink [5], or Colloidal gold [2,6]. Of the protein stains,
Amido black, Ponceau S, and Coomassie blue are the
least sensitive, with a detection limit of approximately
50 ng protein. India ink has an intermediate sensitivity of
detection and takes approximately 4 h to complete. Colloidal gold stain has the highest sensitivity on PVDF blot
membrane (1–2 ng) but requires several hours to complete.
To be eVective, most of the above protein staining procedures are carried out under acidic conditions (e.g., acetic
acid) and/or organic solvents (e.g., methanol). The
eYciency of staining also varied from membrane to membrane. For example, it has been reported that because of
high staining background, nylon membrane is not compatible with all of the above dyes [7]. Furthermore, nitrocellulose membrane cannot be used when high
concentrations of organic solvents such as methanol are
used [7]. The use of acid and/or organic solvents on blots
can also cause problems for the detection of labile antigenic activity, enzymatic activity, protein–protein interactions, and protein–ligand interactions [8].
*
Corresponding author. Fax: +1 215 204 6646.
E-mail address: [email protected] (F.N. Chang).
1
Abbreviations used: PVDF, polyvinylidene diXuoride; BSA, bovine
serum albumin; TTBS, Tris-buVered saline containing Tween 20.
0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2004.11.010
After visualization of protein spots on a blot, it is generally advantageous to remove the dyes from the blot so
that immunoprobing can be carried out with the same
membrane blot. Due to ease of removal, low-sensitivity
dyes such as Ponceau S and Amido black 10B have been
used extensively for this purpose [9]. Such a “reversible
staining” process is diYcult to achieve with the more
sensitive stains such as Colloidal gold, which requires
rather harsh conditions to remove [9]. It is, therefore,
highly desirable to develop a high-eYciency protein
staining method with equal sensitivity toward all blot
membranes that can be carried out under nondenaturing
conditions. To our knowledge, reactive fabric dyes have
not been reported for detecting proteins on membranes.
While assessing various types of reactive fabric dye
stains for their use as a general protein stain, we unexpectedly discovered that Reactive brown 10 (Fig. 1)
stains proteins on blot membranes quickly with a sensitivity of detection comparable to that of Colloidal gold
(1–2 ng). Proteins are visualized within a few seconds in
the staining solution (0.05% in water).
The Reactive brown 10 and other dyes were obtained
from Sigma (St. Louis, MO, USA). Nitrocellulose and
PVDF membranes were obtained from Bio-Rad (Hercules, CA, USA). Nylon membrane was obtained from
Amersham (Buckinghamshire, UK).
PVDF, nitrocellulose, and nylon membrane strips
(1 £ 8 cm) were prepared. Each of the membrane strips
was spotted with 1-l spots of bovine serum albumin
(BSA) in a series of amounts consisting of 1, 2, 5, 50, 100,
500, and 1000 ng of protein. Due to the hydrophobic
nature of the PVDF membrane, the protein solution for
spotting of the PVDF membrane was mixed with an
equal volume of -caprolactone before spotting. After
drying of protein samples, the PVDF membrane was
immersed in methanol (99.9%) brieXy before rinsing with
distilled water for 15 min. Each membrane of each
160
Notes & Tips / Anal. Biochem. 338 (2005) 159–161
Fig. 1. Structure of Reactive brown 10 fabric dye.
composition was then stained with one of a group of Wve
staining compositions selected as follows.
One membrane of each composition was stained
using Ponceau S. In this procedure, the membrane was
incubated in 0.1% (w/v) of Ponceau S stain in 5% acetic
acid for 5 min. The membrane was then destained with
deionized water.
One membrane of each composition was stained
using Amido black 10B. In this procedure, the membrane was incubated in a 0.1% (w/v) solution of Amido
black dissolved in 10% (v/v) acetic acid and 30% (v/v)
methanol. The membrane was incubated for 30 s, and the
stain was removed. The membrane was destained with
deionized water until the background was removed satisfactorily.
One membrane of each composition was stained
using Coomassie blue. In this procedure, the membrane
was incubated in 0.125% Coomassie blue R-250 in 50%
methanol and 10% acetic acid solution for 1 h. The
stain was then removed, and the membrane was
destained with 25% methanol until the background was
removed.
One membrane of each composition was stained
using Colloidal gold. In this procedure, the membrane
was washed three times for 20 min in Tris-buVered
saline, pH 7.4, containing 0.05% Tween 20 (TTBS), followed by three 2-min washes in deionized water. The formulation of Colloidal gold was 5 ml of 1% gold chloride,
6 ml formamide, 5 ml Tween 20, and 3 ml of 0.2 M potassium hydroxide added dropwise, and the total volume
was adjusted to 500 ml with water. The stain was allowed
to stir vigorously overnight, and the pH was brought to
3.5 with formic acid.
One membrane of each composition was stained
using Reactive brown 10 by incubating the membranes
with a 0.05% solution of Reactive brown 10 in distilled
water for approximately 5–10 s. Destaining was carried
out by washing the membrane in water for approximately 30 s.
Fig. 2 shows the sensitivity of all the stain compositions on protein bound to PVDF membrane. It was
noted that both Coomassie blue and Amido black 10B
were diYcult to destain on PVDF membrane and that
high background levels of the stain remained even after
extensive washing. Whereas the sensitivity of detection
for Ponceau S, Amido black 10B, and Coomassie blue is
approximately 50 ng, the sensitivity for Colloidal gold
and Reactive brown is approximately 1 ng.
Fig. 3 shows the sensitivity of all the stain compositions on protein bound to nitrocellulose membrane.
Again, both Colloidal gold and Reactive brown are the
most sensitive, with a detection limit of approximately
1 ng.
Fig. 4 shows the sensitivity of all the stain compositions on protein bound to nylon membrane. None of the
stain compositions, other than Reactive brown 10, was
capable of eVecting protein detection. Reactive brown 10
yielded a reverse staining appearance but nonetheless
gave the same sensitivity on nylon membrane (t1 ng) as
on the other membrane compositions.
Fig. 2. Comparison of the sensitivities of various stains on PVDF
membrane. BSA (1 mg/ml) was prepared as a stock solution, and a
series of dilutions were spotted onto strips of PVDF membrane. Each
membrane was then stained using a diVerent staining method. Lane 1:
membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip
stained with 0.1% Amido black 10B. Lane 3: membrane strip stained
with 0.125% Coomassie blue. Lane 4: membrane strip stained with
Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive
brown 10.
Fig. 3. Comparison of the sensitivities of various stains on nitrocellulose membrane. A series of dilutions were spotted onto strips (as
described previously) of nitrocellulose membrane. Each membrane was
then stained using a diVerent staining method. Lane 1: membrane strip
stained with 0.1% Ponceau S. Lane 2: membrane strip stained with
0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125%
Coomassie blue. Lane 4: membrane strip stained with Colloidal gold.
Lane 5: membrane strip stained with 0.05% Reactive brown 10.
Notes & Tips / Anal. Biochem. 338 (2005) 159–161
161
brown 10 has been described. This procedure takes only
a few seconds to stain and less than 1 min to destain. The
sensitivity of protein staining at approximately 1 ng is
comparable to that of the Colloidal gold method. Unlike
the Colloidal gold method, this method also stains proteins on nylon membranes with the same eYciency as on
other membranes.
References
Fig. 4. Comparison of the sensitivities of various stains on nylon membrane. A series of dilutions were spotted onto strips (as described previously) of nitrocellulose membrane. Each membrane was then stained
using a diVerent staining method. Lane 1: membrane strip stained with
0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido
black 10B. Lane 3: membrane strip stained with 0.125% Coomassie
blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5:
membrane strip stained with 0.05% Reactive brown 10.
In the above studies, BSA was used as the standard
protein. Essentially identical sensitivity toward Reactive
brown 10 dye was observed with other proteins such as
trypsin inhibitor and carbonic anhydrase.
Unlike Colloidal gold staining, the Reactive brown 10
staining and destaining procedures were carried out in
water and not under denaturing conditions (e.g., acidic
and/or organic solvents). This will be of value for preservation of labile antigenic, binding, or catalytic activities
when further characterization is required. In addition,
unlike the Colloidal gold staining, the Reactive brown 10
stain is easily reversible; it can be completely removed by
incubating under alkaline conditions (e.g., 0.1 N NaOH)
for approximately 10 min so that the same blot can then
be used for immunodetection.
A simple procedure for fast and high-eYciency staining of proteins on various membranes using Reactive
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