Protein blotting
Development Technique File No. 220
PhastSystem™
This technique file gives examples of methods for
protein blotting from PhastGel™ media using
diffusion blotting. These examples can serve as a
guide and starting point for your blotting
experiments. This file also contains additional
information to help you choose blotting technique,
transfer milieu and blotting membrane.
Introduction
Protein blotting is the transfer of proteins,
separated by electrophoresis, from a gel to a
membrane. When the protein is bound to a
membrane it is more accessible for specific
identification methods. Identification is easier,
faster and more sensitive on membranes than in
gels. Also, diffusion is reduced when the protein
band is immobilized on a membrane.
Since Southern in 1975 described the transfer of
DNA to nitrocellulose (NC) membranes many
modifications and improvements of the blotting
technique have been published. In 1979 Renhart
(1) adapted blotting to proteins. Today several
different methods for protein blotting are
described in the literature. However, it is
important to choose the appropriate conditions to
obtain optimal results.
Most types of electrophoresis gels are suitable for
blotting. Proteins can be eluted from the gel by
diffusion, by liquid flow (using capillary forces or
vacuum), or by electrophoretic transfer. It is
important to choose a transfer buffer which
promotes optimal elution of proteins out of the gel
and good binding to the membrane.
This file is divided into 2 sections. The first section
discusses blotting techniques, transfer conditions,
membranes and detection. The second section
gives examples of methods for diffusion blotting.
80-1311-99
Edition AB
Choosing the technique
Diffusion blotting
Diffusion blotting is simple to perform and
requires little special equipment. Proteins are
transferred by diffusion from the gel to the
membrane where they are immobilized. PhastGel
media, being ultra-thin, are particularly suitable
for diffusion blotting since the short diffusion
distance results in reduced elution time and sharp
bands. Diffusion blotting from PhastGel media is
performed by simply placing a membrane on the
gel surface followed by incubation at a suitable
temperature. Two examples of methods for
diffusion blotting are given in the method section.
From PhastGel media it takes approximately
20 minutes to obtain good blotting results with
either of the two methods described.
Typical transfer recoveries of protein are about
30–35% after diffusion blotting (2). Since
diffusion is not as efficient as an electrical field in
eluting proteins, it may be difficult to elute very
large proteins e.g. from SDS gradient gels by
diffusion blotting. However, smaller proteins are
easily eluted by diffusion.
Electrophoretic transfer
Electrophoretic transfer is the technique of choice
for many applications since it is rapid and very
efficient, especially when blotting large proteins
from SDS gels for complete transfer of proteins
present in very small amounts. Proteins are eluted
by an electric current passed through the gel at
right angles to the gel surface. To achieve complete
elution from a gel it is important to have a
uniform electric field over the entire gel surface
(3, 4). For electrophoretic transfer, we recommend
PhastTransfer™ for semi-dry blotting of PhastGel
separation media. Detailed methodology is
described in Development Technique File Nos. 221
and 230.
Choosing transfer conditions
Transfer buffer
Low concentrations of non-ionic detergents in the
transfer buffer can also promote elution (3).
Usually the standard buffers work satisfactorily,
but for optimal transfer the conditions may have
to be modified for each case.
It is important to choose conditions which
promote elution from the gel and binding to the
membrane. One can follow the recommendations
given by the membrane manufacturers or use
standard recipes (Table 1) containing Tris/glycine
pH 8.3, with or without additives e.g. methanol or
non-ionic detergents. Both give good results in
most cases. The result, especially with
electrophoretic transfer, will be influenced by the
ionic strength, pH, presence of additives and
temperature.
Temperature
In diffusion blotting the temperature greatly
influences the transfer of proteins. An elevated
temperature increases the diffusion rate. An
example of diffusion blotting at 70 °C is shown in
Figure 1. Note that some native proteins may be
denatured or loose activity at elevated
temperatures.
Table 1. Examples of some commonly used transfer buffers in
relation to type of electrophoresis and type of membrane.
MeOH (methanol). HAc (acetic acid).
Electrophoresis
Buffer
Membrane Reference
SDS, Native
IEF and 2D
25 mM Tris,
192 mM
Glycine, 20%
MeOH
25 mM Tris,
192 mM
Glycine
25 mM Tris
192 mM
Glycine
0.7% HAc
NC
IEF
SDS, Native,
IEF and 2D
Urea, Native,
IEF
6,15
NC
16
Nylon
15
NC, Nylon
6
pH selection
Fig. 1. Example of diffusion blotting at an elevated
temperature (70 °C) after SDS-PAGE on PhastGel gradient
10–15. Transfer to the NC membrane (0.2 µm) was performed
for 20 minutes. Buffer: distilled water. Aurodyne was used to
stain the blotted proteins. The samples are from left to right:
E. coli (lanes 1–4), LMW calibration kit (lanes 5–8).
At high pH most proteins are negatively charged.
A net negative charge is important for rapid
electroelution of the entire protein pattern. Basic
proteins which have a high pI, and are thus poorly
negatively charged or have a net positive charge at
pH 8, can be transferred under acid conditions
(anodal electroelution). Here the positively
charged proteins move from the anode (+) to the
cathode (–) and the gel surface is covered by a
membrane facing the cathode. Negatively charged
proteins can be immobilized on most membranes
while positively charged proteins may bind poorly
to positively charged membranes.
Choosing membranes
Nitrocellulose
Nitrocellulose (NC) is the most commonly used
blotting membrane (see Table 2). It has a proteinbinding capacity of 80 µg/cm2 (2) and is available
in the following pore sizes: 0.1, 0.2 and 0.45 µm.
Binding usually takes place at pH 8 but both basic
and acidic conditions can be used i.e. both
negatively and positively charged proteins can be
immobilized. The binding is mainly due to
hydrophobic interaction but the mechanism is not
fully understood. The amount of proteins bound
to the membrane can be increased by choosing a
membrane of smaller pore size, thus reducing
protein loss through the pores. For electroelution
of small proteins, membranes with 0.1 or 0.2 µm
pores should be chosen. Many general protein
stains (see Table 3) are compatible with NC
membranes.
Additives
Methanol is said to promote binding of proteins to
NC membranes (2). However, due to its fixation of
proteins in the gel, methanol reduces elution
efficiency of large proteins. Inclusion of methanol
improves binding of small proteins to the NC
membranes without significantly affecting their
elution efficiency.
2
Table 2. Blotting membranes and some of their features.
Membrane
Binding capacity
Notes
Nitrocellulose
80 µg
250 µg/cm
Nylon-based
480 µg/cm
215 µg/cm
PVDF
190 µg/cm
Well documented
Pore size is critical
Compatible with anionic stains
Moderate binding
Good binding
Strong
High non-specific binding
Not compatible with anionic stains
Strong
Compatible with anionic stains
Little experience
Nylon-based membranes
NC and PVDF membranes are the best to use with
general protein stains since they do not bind
anionic dyes as nylon membranes do. An
important thing to remember is that the detection
level for most of these stains (Table 3) lies in the
µg range, thus they may not be sensitive enough
for all purposes. On the other hand, detection in
the ng range is possible with Indian Ink and
Aurodye (Janssen Life Science Prod.), Fig. 1.
General protein staining for PhastGel media and
membranes may be performed in the Development
Unit of PhastSystem, see Development Technique
File Nos. 200 and 210.
Specific detection and identification
Table 3. Examples of some commonly used protein stains,
and their compatibility with different blotting membranes.
NC, PVDF
5
By visualizing size/pI markers on the blot with a
general protein stain, it is possible to characterize
a specific protein by size/charge. General protein
staining can be performed after specific protein
identification.
Polyvinyldifluoride (PVDF) has good mechanical
strength and a protein binding capacity similar to
that of NC membranes (5). PVDF immobilizes
proteins by a hydrophobic interaction and the
membrane is compatible with anionic dyes
(Table 3).
Coomassie Brilliant
Blue R-350
Aurodye
Ferridye
Amido Black
Indian Ink
2
5
The success of transfer can be checked by staining
both the gel and the blot with a general protein
stain. For a more precise evaluation of the transfer
a radioactive labelled sample is needed.
Polyvinyldifluoride
Compatible membrane
2
5
Detection
General Protein Staining
Nylon-based membranes, modified with positively
charged groups, have a protein binding capacity of
about 480 µg/cm2 (2). The high binding capacity
of these smooth and mechanically strong
membranes is partly due to the strong electrostatic
interactions between negatively charged proteins
and the positively charged membrane. While a
high binding capacity offers advantages such as
increased sensitivity and the possibility to work
with large amounts of sample, there are
disadvantages. The main disadvantage is the high
non-specific binding of proteins to the membrane;
this results in high backgrounds when probing a
blot unless sufficient blocking of non-specific
binding sites has been carried out. Most general
protein stains cannot be used with nylon
membranes as these membranes bind anionic dyes.
Stain
Reference
Specific interactions such as: antigen-antibody,
receptor-ligand, glycoprotein-lectin, protein-ligand
and DNA-protein allow specific detection and
identification of proteins. Specific detection is
usually simple to perform and very sensitive (ng/pg
range). See method example.
NC, PVDF
NC, Nylon, PVDF
NC, PVDF
NC, PVDF
3
Blocking
Proteins used for specific detection will to some
extent bind non-specifically to the membrane
causing false signals and high background. These
effects are prevented by using another substance to
block the sites which cause non-specific binding.
Some of the most commonly used blocking agents
are listed in Table 4.
Fig. 2. Example of diffusion blotting from PhastGel IEF 3–9
using a NC-membrane. Buffer: 25 mM Tris, 192 mM
Glycine, 20% methanol, pH 8.3. The sample is a dilution
series of transferrin (6 µg→0.05 µg. Left: the gel stained with
PhastGel Blue R. Right: immunoblot of transferrin.
Table 4. Examples of different blocking agents.
Blocking agent
Reference
3%
3–5%
1–10%
10%
5%
5%
10%
0.05%
7
6, 13
14
8
9
10
11
12, 13
Gelatin
BSA
Defatted Dry Milk
Animal Sera
Hemoglobin
Ovalbumin
Ethanolamine
Tween 20
Specific ligand
The antigen-antibody system is the most
commonly used system today. Here an antibody to
the protein of interest is used as the specific
detector. In a direct antibody detection system the
antibody will be either radiolabelled or coupled to
an enzyme of fluorescent tag. The more sensitive
indirect system involves the use of a second
antibody. Here the first specific (often monoclonal)
antibody directed against the protein of interest is
unlabelled and the second antibody (usually
polyclonal), directed against the first antibody, is
labelled.
Fig. 3. Example of diffusion blotting after a native PAGE
separation on PhastGel gradient 8–25. A NC membrane was
used. Buffer: 25 mM Tris, 192 mM glycine, 20% methanol,
pH 8.3. The sample is human plasma, diluted 5 fold. Left: the
gel stained with PhastGel Blue R. Right: immunoblot of C3
and hemopexin.
Blotting from PhastGel IEF and native
gradient gel media
1. Equilibrate the blotting membrane (NC) in
transfer buffer: 25 mM Tris, 192 mM glycine,
and 20% methanol (final pH 8.3) for at least
5 minutes.
2. When the separation is complete, remove the
gel from the separation bed and place it on a
flat surface.
3. Position the equilibrated NC-membrane
carefully on the gel surface and cover with a
Petri dish lid to maintain a humid atmosphere
during the transfer. Note: It is important to
avoid air bubbles between the gel and the
membrane when applying the membrane on
gel. Air bubbles result in poor transfer. Avoid
covering the stacking gel zone with the blotting
membrane otherwise they will stick together.
4. Blot for 10 minutes (Figs. 2 and 3) or longer.
5. Use forceps to remove the NC-membrane
carefully from the gel. Moisten the membrane
with distilled water if necessary.
6. Visualize with a general protein stain or with a
specific detection method (Figs. 1 and 3).
Method examples diffusion blotting
Below we describe two methods for uni-directional
diffusion blotting from PhastGel media. IEF and
native separations (Figs. 2 and 3) are blotted at
room temperature while the SDS separation
(Fig. 4) is blotted at an elevated temperature
(70 °C). The Peltier element of the separation bed
can both cool and heat. Thus, by performing
diffusion blotting on the separation bed, it is
possible to choose any temperature up to 70 °C.
4
Blotting from SDS processed PhastGel
gradient media
After the transfer
1. Block the NC membrane for 30 minutes in
blocking buffer: 20 mM Tris, 500 mM NaCl,
and 3% gelatin.
2. Incubate with the specific antibody for
60 minutes at room temperature. The specific
antibody is diluted with buffer*.
3. Rinse in distilled water.
4. Wash twice in buffer* for 5–10 minutes.
5. Incubate with the secondary antibody for
60 minutes at room temperature. The
secondary antibody is diluted with buffer*.
6. Rinse with distilled water.
7. Wash as in step 4.
8. Peroxidase substrate: Dissolve 10 mg
4-chloro-1-naphtol in 3.3 ml methanol (ice
cold). Add 16.7 ml buffer* containing 10 µl
30% H2O2 (ice cold). Use immediately.
Develop for 15 minutes.
9. Stop the reaction with distilled water.
1. Leave the gel on the separation bed after
electrophoresis, but remove the buffer strip
holder.
2. Set the standby temperature to 70 °C (Fig. 4) or
to other chosen temperature.
3. Dampen a membrane (NC) in distilled water.
Place the pre-wetted membrane on the
separation gel zone (avoid air bubbles). Do not
cover the stacking gel zone with the membrane
otherwise they will stick together.
4. Replace the buffer strip holder (to prevent the
electrodes from touching the gel).
5. Close the lid and blot for 20 minutes (Fig. 4).
or longer.
6. Dampen the membrane with distilled water if it
has dried out after the blotting.
7. Carefully remove the membrane with forceps.
8. Visualize with desired detection method.
References
1. Transfer of proteins from gels to diazobenzyloxymethylpaper and detection with antisera: A method for studying
antibody specificity and antigen structure. Proc. Natl.
Acad. Sci. USA 76 (1979) 3116–3120, Renart, J., et al.
Fig. 4. Example of diffusion blotting after SDS-PAGE
separation on PhastGel gradient 10–15. The transfer to a NC
membrane was performed for 20 minutes at 70 °C. Buffer:
distilled water. The sample is human plasma. Left: the gel
stained with PhastGel Blue R. Right: (from left to right)
immunoblot of albumin, hemopexin, C3, C4 and transferrin.
2. Protein blotting. Electrophoresis 7 (1986) 1–18,
Beisiegel, U.
3. Protein blotting: Principles and applications. Anal.
Biochem. 131 (1983) 1–15, Gershoni, J. M., Palade,
G. E.
4. Protein blotting in uniform or gradient electric fields.
Anal. Biochem. 144 (1985) 32–40, Gershoni, J. M., et al.
Method example specific protein
staining
Horse radish peroxidase
5. Manufacturer’s information.
6. Electrophoretic transfer of proteins from polyacrylamide
gels to nitrocellulose sheets: Procedure and some
applications. Proc. Natl. Acad. Sci. 76 (1979)
4350–4354, Towbin, H., et al.
Specific antigen-antibody detection is a frequently
used method for detecting, identifying and
characterizing proteins and protein–protein
interactions. The scheme below shows a method
for specific identification of proteins, blotted by
diffusion, from IEF, native and SDS separations
(Figs. 2, 3 and 4). In this particular case we
describe the use of NC membranes and a
secondary antibody conjugated with peroxidase
for enzymatic visualization.
7. Improved blocking of non-specific antibody binding sites
on nitrocellulose membranes. Electrophoresis 5 (1984)
54–55, Sarivis, C. A.
8. Identification of Concanavalin A-binding proteins after
sodium dodecyl sulfate-gel electrophoresis and protein
blotting. Anal. Biochem. 123 (1982) 143–146,
Hawkes, R.
9. Shiverer peripheral myelin contains P2. Nature 298
(1982) 471–472, Winter, J.
10. Humoral immune response in human syphilis to
polypeptides of treponema pallidum. J. Immunol. 129
(1982) 1287–1291. Hanff, P. A., et al.
11. Senescent cell antigen is immunologically related to band
3 (membrane proteins/erythrocytes/phagocytosis/aging
protein). Proc. Natl. Acad. Sci. 80 (1983) 1631–1635,
Kay, M. M. B., et al.
* Buffer for IEF: 20 mM Tris, 500 mM NaCl, 0.00125% Tween.
* Buffer for native and SDS-PAGE: as above, without Tween.
5
12. The use of Tween 20 as a blocking agent in the
immunological detection of proteins transferred to
nitrocellulose membranes. J. Immunol. Methods 55
(1982) 297–307, Batteiger, B., et al.
15. Electrophoretic transfer of Proteins from sodium dodecyl
sulfate–polyacrylamide gels to a positively charged
membrane filter. Anal. Biochem. 124 (1982) 396–405,
Gershoni, J. M., Palade, G. E.
13. Effects of the blocking agents bovine serum albumin and
Tween 20 in different buffers on immunoblotting of brain
proteins and marker proteins. J. Immunol. Methods 88
(1986) 233–237, Wedege, E., Svenneby, G.
16. Native protein blotting after isoelectric focusing in fabric
reinforced polyacrylamide gels in carrier ampholyte
generated or immobilized pH gradients. Electrophoresis 9
(1988) 497–511, Klinzkofer-Peresch, A., et al.
14. Immunoblotting with monoclonal antibodies: Importance
of the blocking solution. Anal. Biochem. 159 (1986)
386–389, Hauri, H.-P., Bucher, K.
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