Chapter 11

Multiple Immunoblots by Passive Diffusion

of Proteins from a Single SDS-PAGE Gel
Biji T. Kurien and R. Hal Scofield

Abstract
Western blotting enables the detection and characterization of proteins of low abundance. Sodium dodecyl
sulfate (SDS) polyacrylamide gel-separated proteins are normally transferred electrophoretically to nitro-
cellulose or polyvinylidene difluoride membranes. Here we describe the transfer proteins [Ro 60 (or SSA)
autoantigen, 220 and 240 kDa spectrin antigens, and prestained molecular weight standards] by diffusion
from SDS polyacrylamide gels at 37 °C. Up to 12 immunoblots can be obtained from a single gel by this
method.

Key words Non-electrophoretic transfer, Immunoblots, Bidirectional transfer, Nitrocellulose mem-

brane, Autoantibodies, Autoantigens

1 Introduction

Western blotting, involving electrophoretic transfer of proteins to

a microporous membrane support with subsequent immunodetec-
tion [1], has made a tremendous impact in the field of immunology.
Proteins separated on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) are transferred electrophoretically to
mainly nitrocellulose or polyvinylidene difluoride [1–4]. Protein
transfer from gels to membranes has been achieved in three ways:
(a) simple diffusion [3], (b) vacuum-assisted solvent flow [5, 6],
and (c) electrophoretic elution [1, 7, 8]. Only electrophoretic elu-
tion has been used widely owing to a variety of reasons, including
efficiency, simplicity, and length of transfer. Bidirectional transfer
procedure was demonstrated in 1980, when Smith and Summers
[9] transferred DNA and RNA from polyacrylamide gels to nitro-
cellulose in 36 h to obtain two blots. Here we demonstrate that
proteins can be efficiently transferred non-electrophoretically from
SDS-PAGE gels to nitrocellulose membranes. A similar procedure
has been used in 1982 [10] to transfer proteins from thin (0.5 mm)

Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology,
vol. 1312, DOI 10.1007/978-1-4939-2694-7_11, © Springer Science+Business Media New York 2015

77
78 Biji T. Kurien and R. Hal Scofield

native isoelectric focusing gels to nitrocellulose membranes to obtain

two blots in 1 h. Diffusion-mediated transfer of immunoglobulins
from one side of a native gel after isoelectric focusing to an anti-
gen-coated nitrocellulose sheet has been achieved in a similar
fashion [11, 12]. We were able to transfer and immunologically
detect a 60,000 molecular weight autoantigen (Ro 60) (Fig. 2) as
well as the spectrin antigens (molecular weight >200,000) (Fig. 3)
using this method. When prestained molecular weight standards
were transferred, only markers up to 118,000 molecular weight
could be visualized (these proteins were not visualized immuno-
logically) (Figs. 2 and 3).
Thus, we have obtained up to 12 immunoblots from a single
gel using multiple antigens (high-, intermediate-, and low-
molecular-weight proteins) and multiple sera. Subsequently several
investigators have also obtained similar results and also in a quan-
titative manner (see Chapters 9 and 10).

2 Materials

Prepare all solutions using ultrapure water (prepared by purifying

deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations when disposing waste materials. We do
not add sodium azide to reagents.

2.1 SDS 1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Add about
Polyacrylamide Gel 100 mL water to a 1 L graduated cylinder or a glass beaker (see
Note 1). Weigh 181.7 g Tris–HCl and transfer to the cylinder.
Add water to a volume of 900 mL. Mix and adjust pH with
HCl (see Note 2). Make up to 1 L with water. Store at 4 °C.
2. Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Weigh 60.6 g
Tris–HCl and prepare a 1 L solution as in previous step. Store
at 4 °C.
3. Thirty percent acrylamide/Bis solution (29.2:0.8)
acrylamide:bis: Weigh 29.2 g of acrylamide monomer and
0.8 g Bis (cross-linker) and transfer to a 100 mL graduated
cylinder containing about 40 mL of water. Add a spatula of AG
501-X8 (D) mixed-resin beads and mix for about 30 min.
Make up to 100 mL with water and filter through a 0.45 μm
Corning filter (see Note 3). Store at 4 °C, in a bottle wrapped
with aluminum foil (see Note 4).
4. Ammonium persulfate: 10 % solution in water (see Note 5).
5. N,N,N,N′-Tetramethyl-ethylenediamine: Store at 4 °C
(see Note 6).
 Non-Eectrophoretic Bi-Directional Transfer to Membranes 79

6. SDS-PAGE running buffer: 0.025 M Tris–HCl, pH 8.3,

0.192 M glycine, 0.1 % SDS (see Note 7).
7. SDS lysis buffer (5×): 0.3 M Tris–HCl (pH 6.8), 10 % SDS,
25 % β-mercaptoethanol, 0.1 % bromophenol blue, 45 % glyc-
erol. Leave one aliquot at 4 °C for current use and store the
remaining aliquots at −20 °C (see Note 8).
8. Bromophenol blue (BPB) solution: Dissolve 0.1 g BPB in
100 mL water.

2.2 Immunoblotting 1. Nitrocellulose membranes.

2. Western blot transfer buffer: 0.025 M Tris–HCl, 0.192 M
glycine, 20 % methanol (see Note 9).
3. Tris-buffered saline (TBS; 10×): 1.5 M NaCl, 0.1 M Tris–HCl,
pH 7.4.
4. TBS containing 0.05 % Tween-20 (TBST).
5. Blocking solution: 5 % milk in TBS (see Note 10). Store at
4 °C.
6. Diluent solution: 5 % milk in TBST (see Note 10). Store at
4 °C.
7. Mini PROTEAN® 3 System glass plates.
8. Medium binder clips (1¼ in.).
9. Plastic container.
10. Wypall X-60-reinforced paper.
11. Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl
phosphate (BCIP): Dissolve 1 g NBT in 20 mL of 70 %
dimethylformamide (DMF). Dissolve 1 g BCIP in 20 mL of
100 % DMF. Add 33 μL of BCIP and 66 μL of NBT to 10 mL
of alkaline phosphatase buffer just before adding to membrane.
Alternatively, use 1-Step™ NBT/BCIP ready-made mix.

2.3 Antigens 1. Purified red blood cell spectrin, anti-spectrin polyclonal anti-
and Conjugates body, anti-hemoglobin antibody.
2. BenchMark prestained molecular weight standards.
3. Purified bovine Ro 60: Purify Ro 60 as reported [13, 14] or
purchase from Immunovision, Springdale, AK, USA.
4. Prepare human erythrocyte membranes according to Dodge
et al. [15].

3 Methods

Carry out all procedures at room temperature unless otherwise

specified.
80 Biji T. Kurien and R. Hal Scofield

3.1 10 % Sodium 1. Mix 2.5 mL of resolving buffer, 3.33 mL of acrylamide

Dodecyl Sulfate- mixture, and 4 mL water in a 50 mL conical flask. Degas
Polyacrylamide Gel with helium for 10 min. Add 100 μL of SDS, 80 μL of ammo-
Electrophoresis nium persulfate, and 10 μL of TEMED and cast gel within a
7.25 cm × 10 cm × 1.5 mm gel cassette. Allow space for stacking
gel and gently overlay with isobutanol or water (see Note 11).
2. Prepare the stacking gel by mixing 1.25 mL of resolving buf-
fer, 0.67 mL of acrylamide mixture, and 3 mL water in a 50 mL
conical flask. Degas with helium for 10 min. Add 100 μL of
SDS, 40 μL of ammonium persulfate, and 5 μL of TEMED.
Insert a 10-well gel comb immediately without introducing air
bubbles.
3. Heat aliquots of bovine Ro 60, RBC membranes, and human
spectrin antigens at 95 °C for 5 min. Do not add lysis buffer to
the prestained protein standard or subject it to heat. Centrifuge
the heated samples at 3,000 × g for 30 s to bring down the
condensate. Load increasing amounts of Ro antigen (1–4 μg)
on one gel and same amounts of spectrin (3 μg/lane) or RBC
membrane antigens on two other gels along with protein stan-
dards (10 μL/well-2 μg/marker/lane). Add protein standards
in every other lane (alternating with spectrin) in the gel with
spectrin. Electrophorese at 15 mA until the sample has entered
the gel and then continue at 25 mA till the dye front (from
the BPB dye in the samples) reached the bottom of the gel
(see Note 12).
4. Following electrophoresis, pry the gel plates open with the use
of a spatula. The gel remains on one of the glass plates. Rinse
the gel with water and transfer carefully to a container with
western blot transfer buffer.
5. Cut a nitrocellulose membrane to the size of the gel and
immerse in methanol. Rinse twice in distilled water and once
with transfer buffer.

3.2 Non- 1. Immediately following SDS-PAGE, when the dye front reaches
electrophoretic the end of the gel, turn off the power supply. Separate the gel
Transfer plates with the help of a spatula or a similar tool. Remove the
stacking gel.
2. Rinse the gel (still supported by the bottom glass plate) care-
fully with deionized water to remove traces of SDS-PAGE run-
ning buffer.
3. Excise the gel with spectrin antigen such that there is one lane
with the protein markers and one with the spectrin antigen.
4. Leave the gels to air-dry for 5–10 min (see Note 13).
5. Gently lay one nitrocellulose membrane, cut to the shape of
the gel, on top of the gel (see Note 14).
 Non-Eectrophoretic Bi-Directional Transfer to Membranes 81

Fig. 1 Gel-membrane assembly for the non-electrophoretic transfer of proteins

from SDS-PAGE gels to nitrocellulose membranes to obtain up to 12 blots. The
polyacrylamide gel is sandwiched between two membranes, filter paper, and
glass plates and incubated at 37 °C for varying periods of time to obtain up to 12
blots (reproduced from ref. 2 with permission from Elsevier)

6. Gently lift the gel-membrane sandwich from the glass plate

and place it on a Whatman No. 3 filter (place membrane side
directly on the filter paper and the exposed gel side on top) cut
to the size of the gel.
7. Place a second nitrocellulose membrane, cut to the shape of
the gel, on top of the gel, followed by a Whatman No. 3 filter
paper cut similarly (see Note 14).
8. Place the nitrocellulose-gel-filter paper sandwich between two
mini-PROTEAN® 3 System glass plates and secure with clamps.
9. Place this assembly in a pre-warmed humidified plastic con-
tainer (Fig. 1) and incubate at 37 °C for 30 min (see Note 15).
Remove the membranes for immunoblotting (see Note 16)
(Fig. 2).
10. Repeat this procedure with another set of nitrocellulose mem-
branes and incubate the assembly at 37 °C for 2 h to obtain
two more blots from the same gel (see Note 17).
11. Use the same gel further to obtain blots by repeating this pro-
cedure to obtain a total of 12 blots. Incubate gel with the
respective membranes for a period of 3 or 4 h to obtain the
third and fourth sets of blots. Obtain the fifth and sixth sets of
blots (Fig. 3) by incubating with the respective membranes for
a period of 9 h or 36 h, respectively (see Note 18).
12. Cut excess membrane to smoothen edges and also cut the spectrin-
containing membrane into individual lanes (see Note 19).
13. Block the membranes with blocking solution for 1 h.
82 Biji T. Kurien and R. Hal Scofield

Fig. 2 Ro 60 immunoblots obtained following non-electrophoretic transfer for

30 min or 2 h. (a) One of the two Ro 60 blots obtained by the first incubation of
nitrocellulose membranes on either side of the gel at 37 °C for 30 min. (b) A blot
from the second set (blots 3 and 4) obtained from the same gel following incuba-
tion at 37 °C for 2 h (reproduced from ref. 6 with permission from Elsevier)

Fig. 3 Immunoblots obtained using spectrin as antigen. Lane 1 in (a)–(f) shows prestained SDS-PAGE molecu-
lar weight standards. Lane 2 shows spectrin probed with either preimmune (b, c, f) or anti-spectrin rabbit sera
(a, d, e). Panel (a) shows one of the first two blots obtained and has been probed with anti-spectrin. Panel (b)
shows the second of the two blots of the first set obtained from the reverse side of the gel and was probed
with preimmune sera. Panels (c) and (d) show the fifth set (blots 9 and 10) of immunoblots probed with preim-
mune and anti-spectrin sera, respectively. Panels (e) and (f) show the sixth set (blots 11 and 12) of blots probed
with anti-spectrin and preimmune sera, respectively (reproduced from ref. 6 with permission from Elsevier)

14. Add appropriate anti-sera to the membranes (anti-spectrin,

anti-Ro 60, or control sera) and incubate for 2 h.
15. Rinse membrane strips with deionized water 2–3 times
(see Note 20).
16. Wash 5× with TBST, 5 min each time.
17. Add anti-human IgG or anti-rabbit IgG alkaline phosphatase
conjugate (1:5,000 dilution, diluted in diluent) and incubate
for 1 h.
18. Wash as in step 14.
 Non-Eectrophoretic Bi-Directional Transfer to Membranes 83

19. Add 500 μL of NBT/5-bromo-4-chloro-3-indolyl phosphate

substrate and let bands develop.
20. Rinse 2–3 times with deionized water (see Note 20).
21. Wash with TBST and arrange strips on paperboard inserts
(see Note 21).

4 Notes

1. Having water at the bottom of the cylinder helps to dissolve

the Tris relatively easily, allowing the magnetic stir bar to go to
work immediately. If using a glass beaker, the Tris can be dis-
solved faster if the water is warmed to about 37 °C. However,
the downside is that care should be taken to bring the solution
to room temperature before adjusting pH.
2. Concentrated HCl (12 N) can be used at first to narrow the
gap from the starting pH to the required pH. From then on it
would be better to use a series of HCl (e.g., 6 and 1 N) with
lower ionic strengths to avoid a sudden drop in pH below the
required pH.
3. Wear a mask when weighing acrylamide. To avoid exposing
acrylamide to co-workers, cover the weigh boat containing the
weighed acrylamide with another weigh boat (similar size to
the original weigh boat containing the weighed acrylamide)
when transporting it to the fume hood. Transfer the weighed
acrylamide to the cylinder inside the fume hood and mix on a
stirrer placed inside the hood. Unpolymerized acrylamide is a
neurotoxin and care should be exercised to avoid skin contact.
Mixed resin AG 501-X8 (D) (anion- and cation-exchange
resin) is used when acrylamide solution is made, since it
removes charged ions (e.g., free radicals) and allows longer
storage. Some investigators store the prepared acrylamide
along with this resin in the refrigerator. However, we filter
them out before storage. The used mixed resin should be dis-
posed as hazardous waste. Manufacturer’s warning states that
this resin is explosive when mixed with oxidizing substances.
The resin contains a dye that changes from blue-green to gold
when the exchange capacity is exhausted.
4. The acrylamide solution can be stored at 4 °C for 1 month.
Acrylamide hydrolyzes to acrylic acid and ammonia. The acryl-
amide mixture, buffer, and water can be prepared in large
batches, frozen in aliquots (for greater day-to-day reproduc-
ibility), and used indefinitely (see ref. 16). Remove the required
amount, bring to room temperature, and add the other ingre-
dients for polymerization. However, in our laboratory we
make the acrylamide solution fresh about every month when
we cast our own gels.
84 Biji T. Kurien and R. Hal Scofield

5. We find that it is best to prepare this fresh each time.

6. We find that storing at 4 °C reduces its pungent smell.
7. Simple method of preparing running buffer: Prepare 10×
native buffer (0.25 M Tris–HCl, 1.92 M glycine). Weigh
30.3 g Tris–HCl and 144 g glycine, mix, and make it to 1 L
with water. Dilute 100 mL of 10× native buffer to 990 mL
with water and add 10 mL of 10 % SDS. Care should be taken
to add SDS solution last, since it makes bubbles.
8. SDS precipitates at 4 °C. Therefore, the lysis buffer needs to be
warmed prior to use.
9. Dilute 100 mL of 10× native buffer to 800 mL with water and
add 200 mL of methanol. Avoid adding methanol directly to
the 10× buffer, since it precipitates its ingredients. Even in
such a scenario the precipitate can be redissolved by the addi-
tion of 800 mL water.
10. Add 100 mL of 10× TBS to a 1 L graduated cylinder and make
it to about 800 mL with water. Transfer 50 g skim milk pow-
der into the cylinder and stir until dissolved. Make to 1 L with
water. Separate 500 mL as the blocking solution. To the
remaining 500 mL add 250 μL of Tween-20 (cut end of blue
tip to aspirate Tween-20 easily), dissolve, and use it as the
diluent.
11. The gel cassette was sealed at the base using 1 % agarose.
Overlay the resolving gel with water for gels having acrylamide
concentration lower than 8 % and use isobutanol (or isobutanol
saturated with water) for gels of 10 % or greater (see ref. 17).
This overlay prevents contact with atmospheric oxygen (which
inhibits acrylamide polymerization) in addition to helping to
level the resolving gel solution.
12. Centrifuging the samples prior to the run helps remove insol-
uble debris, which could produce streaks in the protein lanes
(revealed when stained with Coomassie blue). Add a drop of
0.1 % BPB to the upper chamber buffer. This helps to form a
much stronger dye front during the electrophoretic run.
13. Membrane contact with the gel is much better when the gel is
not moist. Therefore it is important to dry the gel for 5–10 min.
The membrane will now stick well to the gel and gel will peel
off the bottom glass plate by just lifting the membrane.
14. Hold the two top corners of the membranes with each hand.
Lower the bottom part of the membrane first on the lower
part of the gel and gently release the membrane little by little
to lay the complete membrane on the gel. This will prevent
trapping of bubbles in between the gel and the membrane.
A 10 mL pipette was used to roll out air bubbles from the
 Non-Eectrophoretic Bi-Directional Transfer to Membranes 85

gel-membrane sandwich prior to placing in transfer cassette.

In the case of the gel with spectrin, cut the membrane to fit
the two lanes of the gel.
15. The humid chamber consisted of a closed plastic container
with a moist Terri Wipes paper towel at the bottom. The con-
tainer must be big enough to contain the nitrocellulose-gel-filter
paper assembly encased within the glass plates.
16. The second set of two blots was also obtained following incu-
bation with the gel for 1 h (see Fig. 3b, c).
17. While removing the nitrocellulose membranes from the gel for
immunoblotting, it would be common to find that the gel
comes up stuck to one of the two membranes. To remove this
membrane from the gel, place a fresh, dry nitrocellulose mem-
brane on top of the gel and gently lift the gel. The gel becomes
stuck to this fresh membrane, thus releasing the other
membrane.
18. Gel dries, in spite of placing in humid chamber, when incu-
bated for longer time periods (36 h). Therefore it is best to use
the blots obtained after 12-h incubation with the membrane.
19. Cut a tiny wedge from the bottom left side of the marker lane
and the main membrane sheet for orientation purposes. Also,
in the case of the membranes with spectrin (see Subheading 3.1,
step 3), excise the spectrin lane from the protein marker lane
after matching each spectrin lane with its specific protein
marker lane with pencil marks.
20. Rinsing the membrane strips with deionized water 2–3 times
will help remove a bulk of the nonspecific antibodies and help
reduce the amount of TBST used subsequently and also reduce
the number of washes. This wash helps to reduce nonspecific
binding of NBT/BCIP to the strip. The water, owing to its
low ionic strength compared to TBST, will be able to remove
contaminants much better than TBST. Water is much cheaper
compared to TBST, in terms of money and labor. Other inves-
tigators have found no reduction in detection of specific sig-
nals due to washing with water [18].
21. We use paperboards placed in between stacks of ELISA plates
in packages of ELISA plates (Costar, Cambridge, MA, USA)
for this purpose.

Acknowledgement

This work was supported by NIH grant ARO1844 and Oklahoma

Center for the Advancement of Science and Technology to R.H.S.
86 Biji T. Kurien and R. Hal Scofield

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