Western Blotting: Advanced article

Immunoblotting . Principles
Article Contents

Ole J Bjerrum, University of Copenhagen, Denmark . Protein Blotting Techniques

. Specificity Problems
Niels HH Heegaard, Statens Serum Institut, Copenhagen, Denmark . Applications

. Limitations
Western blotting is a method by which proteins that have been physically separated and Online posting date: 15th March 2009
subsequently immobilized on the surface of a membrane are probed for reactivity with
different types of affinity reagents, such as antibodies, receptors, are tested for the
presence of interacting proteins in pure preparations or complex mixtures.
Immunoblotting and the major steps of the procedure are described. It concerns the
most commonly applied separation technique: sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDSPAGE); the major transfer techniques: semidry or tank blotting;
the many protein stains where the choice is depending on the sensitivity wanted and
selection of the right blocking procedure. Visualization of bound probes represents a
major issue as the variations are legio, but where the use of enzyme conjugated
antibodies for colorimetric, chemo- and bioluminiscence is the most applied ones. The
limits for quantitative use of the technique, the specificity problem crucial for a proper
interpretation and various application, are described.

Principles Electrophoresis: One-dimensional; Gel Electrophoresis of

Proteins: High-resolution Two-dimensional
Blotting is the process of transferring physically separated
analytes from a separation matrix to the surface of an im-
mobilizing membrane. The transfer step is preceded by Protein Blotting Techniques
one- or two-dimensional separation procedures and fol-
lowed by a detection step that benefits from the increased The standard approach for protein immunoblotting in-
accessibility of the transferred molecules on the blotting volves a sodium dodecyl sulfate–polyacrylamide gel elect-
membrane surface. When antibodies are used as probes, rophoresis (SDS-PAGE) separation electrotransferred to
the procedure is called immunoblotting. Figure 1 shows the nitrocellulose or polyvinylidene difluoride (PVDF) mem-
principle of electroimmunoblotting. If no separation is in- branes. Alternatives that are useful in specific cases will be
volved in the primary step or there is no contact between mentioned below.
the separation medium and the blotting membrane, e.g.
when liquid chromatography fractions are applied to the The separation step
membrane, the approach is often called dot, slot or line- Any protein separation method that leaves the separated
blotting or – when antibodies are involved – immuno- analytes in a matrix on which a blotting membrane can be
binding. Several recent, comprehensive reviews cover the placed and through which a fluid flow can pass may be used
majority of options including instruments and step-by-step to provide the initial separation step. Proteins separated by
recipes (Dunbar, 1994; Heegaard and Bjerrum, 1988). We one- or two-dimensional gel electrophoresis are routinely
here focus on immunoblotting techniques. See also: Gel blotted (Egger and Bienz, 1994; Dunn, 1999), but molec-
ular patterns from other separation methods such as thin-
layer chromatography can also be blotted (Taki et al.,
1994) and fractions can be dot blotted from liquid chro-
matography or capillary electrophoresis (Konse et al.,
ELS subject area: Genetics and Molecular Biology 1993). See also: Chromatographic Techniques
How to cite: Transfer techniques and choice of blotting
Bjerrum, Ole J; and, Heegaard, Niels HH (March 2009) Western Blotting:
Immunoblotting. In: Encyclopedia of Life Sciences (ELS). John Wiley & membranes
Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0000995.pub2
Transfer onto the membrane may be accomplished by mo-
lecular diffusion, by a buffer flow induced by suction

ENCYCLOPEDIA OF LIFE SCIENCES # 2009, John Wiley & Sons, Ltd. www.els.net 1
 Western Blotting: Immunoblotting

Figure 1 General principle of electroimmunoblotting. Protein bands in a gel are electrotransferred to a membrane and subsequently probed with primary
antibodies. After washing, the bound primary antibodies are detected by means of enzyme-conjugated secondary antibodies that cause the development of
precipitating colour after addition of the appropriate substrate. Modified from Bjerrum OJ and Heegaard NHH (eds) (1988) Handbook of Immunoblotting of
Proteins, vol. I, p. 3. Copyright CRC Press, Boca Raton, FL, 1988.

(microfiltration) or capillary action, or most often used by quantitatively and qualitatively faithful image of the orig-
electrotransfer where charged molecules are transferred to inal separation pattern with only minute amounts of pro-
the membrane by electrophoresis. tein remaining in the original gel. To ensure efficient
Diffusion is simple but too inefficient to apply for most transfer a good control is to always stain the gel for protein
transfers. From agarose gels transfer is easily achieved by a after the transfer step. For thin-layer chromatography
buffer flow, but precipitated proteins in immunoelectro- (TLC) blotting the transfer takes place by placing a 2008C
phoresis patterns must be dissociated prior to mobilization hot iron on a wetted PVDF membrane placed on the TLC
(Bjerrum et al., 1987). Proteins from isoelectric focusing plate (Taki et al., 1994). See also: Proteins: Fundamental
gels may be electrotransferred after addition of SDS (Stott, Chemical Properties
1989). Proteins separated in polyacrylamide gels are most The two types of membranes most often used are nitro-
often electroblotted. This usually works very well and takes cellulose and PVDF, which display only small differences
place either in solution or wet transfer (tank blotting) or in with respect to binding capacity and staining compatibility
a semidry buffer system using wetted filter papers sand- (Egger and Bienz, 1994). Their protein-binding capacity
wiched between planar electrodes. In the first case gradient appears to be around 100–200 mg cm22 and both may be
electric fields may make elution rates more uniform along stored dry at 2208C or 48C in sealed plastic bags for a
the gel (Gershoni, 1988). However, the semidry approach is prolonged period (a year) after transfer. The strength of
now widely used. protein binding to the membrane is an important variable
As transfer buffers, diluted SDS-PAGE running buffers in different applications. The use of membranes that binds
will often suffice (Heegaard and Bjerrum, 1988). Proteins less tightly is warranted for preparative applications and
are transferred toward the anode because of the anionic also gives a lower background in immunoblotting. PVDF
SDS and the slightly alkaline pH of the usual transfer membranes exist in different pore size versions designed for
buffers. Too much SDS may block membrane binding these applications (LeGendre, 1990). They have to be wet-
of proteins but 0.01% facilitates the transfer, especially of ted before use, e.g. in methanol, but are otherwise easy to
larger proteins. Addition of methanol up to 20% counter- handle and have a high chemical resistance. The main dis-
acts swelling of the gel and increases the binding of proteins advantage of nitrocellulose is its low mechanical strength
(especially small ones) to nitrocellulose; however, it when dry (reinforced versions do exist), less binding
also impedes transfer and concentrations should therefore of small proteins and peptides than PVDF and incompat-
be balanced for this. Highly crosslinked and thick polya- ibility with many organic solvents. However, nitrocellulose
crylamide gels and hydrophobic and large molecules membranes are relatively cheap, protein binding is less
take longer to elute whereas, conversely, small molecules sensitive to the presence of SDS than is the case for PVDF
transferred to large pore size blotting membranes may and background staining in immunodetection is less than
pass through the membrane if there is a local saturation of when using high-retention PVDF membranes (Ursitti
the protein-binding capacity. For these reasons current et al., 1995). Nitrocellulose also comes in different pore
density and transfer time should be balanced. After sizes and the larger pore size generally retains small pro-
adjusting parameters the ideal replica should be a teins less well. Fixation of the blotted proteins to

2 ENCYCLOPEDIA OF LIFE SCIENCES # 2009, John Wiley & Sons, Ltd. www.els.net
 Western Blotting: Immunoblotting

nitrocellulose may in special cases lead to improved reten- recommended for routine use. Dichromatic and trichro-
tion and detection. Special membranes like Paal FluoroT- matic approaches combine general protein detection with
rans have been developed with fluorescent-based detection specific (e.g. antibody-mediated) detection and are
(Koticha et al., 2006). described below.

General protein staining Blocking, probing and visualization of bound

probes
The extent of transfer can be monitored by staining the
blot, or a part of it, with one of several general protein After transfer, but before probing with antibodies or other
stains. Radioactively labelled proteins can be visualized protein-specific reagents, the surplus of protein-binding
directly by autoradiography and the film subsequently sites on the membranes must be blocked to avoid nonspe-
compared with the blot. In other cases, a general protein cific binding in later steps. The many blocking procedures
stain makes it easier to align the immunovisualized bands that are used reflect the need to optimize this step in each
with the general protein separation pattern (Eynard and individual application. Much used are 1–5% (w/v) solu-
Laurière, 1998). Also, prestained molecular weight mark- tions of proteins such as bovine serum albumin, nonfat dry
ers transferred from the separation gel give a rough im- milk, casein, ovalbumin or gelatin that should not react in
pression of the transfer efficiency and make it easy to orient the antibody steps and should not contain activities that
the blot. General protein staining with Ponceau S, Amido will act on or inhibit the visualization of enzyme substrate.
black, Coomassie blue, organic copper, colloidal gold or Dried milk powder is a cheap and effective blocking agent
silver (in order of increasing sensitivity) is performed before at 0.5–5% for 30 min (Egger and Bienz, 1994), but
the membrane is blocked with solutions of proteins (Egger secondary detection antibodies (e.g. from goat) may
and Bienz, 1994). All general staining techniques are prone cross-react with the milk immunoglobulins and cause high
to interference with subsequent immunodetection of the background. The nonionic detergent Tween 20 is also a fast
stained proteins; however, staining with organic copper is and simple blocking reagent (2% Tween 20 for 2 min will
reversible on both nitrocellulose and PVDF membranes block nitrocellulose). Detergent may increase renaturation
and is compatible with subsequent immunostaining on the blot but may also elute some protein from the blot,
(Antharavally et al., 2004). Procedures for Coomassie or especially if recommended blocking times are exceeded or if
silver staining of proteins after immunostaining on blots other more displacing nonionic detergents such as Triton
have also been described. These methods take advantage X-100 or Nonidet P-40 are used (Bjerrum et al., 1987; Stott,
of using Tween 20 for all blocking and incubation steps 1989). General protein stains cannot be used to detect
(Houen et al., 1997; Sorensen et al., 2002). By far the most blotted bands after blocking with protein. In all cases the
sensitive general protein stains (2 ng protein loaded on the inclusion of 0.01–0.05% Tween 20 in incubation and
gel) are the colloidal metal stains (gold or silver). These are washing buffers is recommended to suppress unwanted
quite simple to use and are compatible with nitrocellulose background binding.
and PVDF membranes. Ponceau S is the least sensitive of Probing with antibodies takes place after the blocking
the general protein stains (about 100 times less sensitive step. Indirect methods are normally used in immunodetec-
than colloidal gold staining) but has the advantages of cost, tion protocols for reasons of sensitivity and simplicity. The
simplicity and of being destainable in plain water. Amido primary antibody, which is unlabelled, is detected by means
black also works on nitrocellulose, PVDF and nylon and of a secondary labelled antibody that reacts with the mol-
has a sensitivity comparable to that of Coomassie (450 ng ecules that remain bound after the first antibody incuba-
band21) but stains faster and under milder conditions than tion (cf. Figure 1). Thus, patient sera or animal immune sera
Coomassie. Neither Coomassie blue, Ponceau S, nor in appropriate dilutions can be used as probing reagents
Amido black interferes with sequencing reactions. A band directly followed by visualization by a broadly reacting
detectable by Coomassie normally contains enough pro- secondary reagent, usually specific for g-globulin class.
tein for sequencing (Matsudaira, 1993). An imaging Since antigen–antibody reactions are involved, the incu-
method unique for PVDF is to wet the membrane in meth- bation and washing buffers are usually physiological with
anol and transilluminate it (LeGendre, 1990). Prolonged respect to pH and salt content. To repel nonspecific bind-
time in methanol or other organics causes the proteins to ing, the pH and salt concentrations in the incubation buff-
elute. Other general protein detection approaches rely on ers can be adjusted so that only strongly binding antibodies
chemical derivatization of the proteins with primary amino react (Heegaard and Bjerrum, 1988). When biotinylated
group-reactive chemicals such as N-hydroxysuccinimide reagents are used, the high-affinity interaction of avidin or
biotin, dinitrophenol or pyridoxal-5¢-phosphate/sodium streptavidin with biotin ensures that procedures using
borohydride (Kittler et al., 1984) or the generation of streptavidin-conjugated enzymatic detection have a high
fluorescent protein-dye adducts on PVDF membranes sensitivity (Dunbar, 1994). However, tissue extracts may
(Daban, 2001). These subsequently can be detected by contain significant levels of endogenous biotin-containing
streptavidin–enzyme conjugates or by specific antihapten proteins and this may disturb the interpretation of blots
antibodies and secondary visualizing agents, as in conven- developed on the basis of streptavidin/avidin-conjugated
tional immunoblotting. These multistep methods are not reagents. Control blots developed without primary

ENCYCLOPEDIA OF LIFE SCIENCES # 2009, John Wiley & Sons, Ltd. www.els.net 3
 Western Blotting: Immunoblotting

reagents show reactivity in these cases and alternative antibody makes trichromatic detection possible using ul-
amplification systems, e.g. based on polymeric enzyme traviolet (UV)-illuminators, laser-based gel scanners or
conjugates with secondary antibodies on dextran back- charge-coupled device (CDD)-cameras. Fluorescent stains
bones may be applicable (Banks et al., 2003). See also: provide greater linear dynamic range for quantification
Antigen–Antibody Complexes; pH and Buffers (Koticha et al., 2006). Recently, use of fluorescent semi-
Many antibody-based detection systems have been conductor nano crystals or quantum dots have also been
developed to identify specific proteins, i.e. by colori- applied (McDonald et al., 2006).
metric, colloidal gold, chemiluminiscence, autoradigraph-
ic, luminiscence and fluorescent detection. The enzymes Quantitation
conjugated with the secondary reagents used for colori-
metric detection are most often alkaline phosphatase (AP) Immunoblotting is at best a semiquantitative technique
or horseradish peroxidase (HRP). The ensuing staining because of the numerous steps involved in the procedure
reaction for enzyme activity is based on precipitating before an antigen meets its antibody. Recovery of antigenic
stains. With HRP the 4-chloronaphthol or 3-amino-9-ethyl material can be highly variable at each of the steps involved
carbazole coupling salts with hydrogen peroxide are widely in the blotting procedure. In addition to quantitative
used, whereas AP staining often uses 5-bromo-4-chloro-3- losses, some or all of the antibody reactivity of the antigen
indolyl phosphate and Nitroblue tetrazolium to effect may be lost during the process. Thus, for quantitation in
staining. The AP-based stains appear to be a little more different blotting systems one must always take the vul-
sensitive and durable than the HRP stains. When a blot is nerability of the specific reagent for alterations of the an-
sealed behind plastic and kept in a binder, the AP staining tigen into consideration. In spite of this, direct visually
appears unchanged after 25 years of storage. The chromo- observed differences reflect quantitative differences of blot-
gens also differ with respect to their health hazards. En- ted antigens and blots visualized on photographic film can
zyme-based staining of bound antibodies has detection be quantitated by scanning with transmission densitome-
limits in the range 0.01–1 ng protein with the lower limits ters, whereas coloured bands must be scanned by reflect-
obtainable with AP-based methods and the higher limits ance densitometry or after making the membrane
seen with HRP. transparent in oil or other compatible organic solvent
Colloidal gold-labelled antibodies give a detection limit (Stott, 1989). Dot blots visualized by chemiluminescence
in the same range as HRP-based systems, but this can be can be densitometrically scanned and the linear range
improved with a silver enhancement step to get to the range identified. In the case of immunoglobulins a detection limit
of AP stains. In chemiluminescence methods enzymatic of 0.05 ng spot21 and a linear response covering two orders
activity is converted to light, which is recorded on a pho- of magnitude were demonstrated (Sulimenko and Draber,
tographic film (Durrant, 1990). Radioactive detection and 2004). Also, it has been shown that common flat bed scan-
chemiluminescence methods offer the most sensitive visu- ners may be used in combination with simple image anal-
alization by being 100–500 times more sensitive than ysis programs to achieve quantitative pixel analysis in both
colorimetric stains (Dakessio and Ashley, 1992). Chemi- dot blots and immunoblots (Vierck et al., 2000). Useful
luminescence from HRP- or AP-based systems emits hints and the superiority of using the reflectance mode of
enough light to secure results in extremely short times (ex- scanning over the transmission mode can be found in
posure times of seconds to minutes). Since light persists for (Tarlton and Knight, 1996).
hours, multiple exposures are possible. Substituted 1,2-di-
oxetane phosphates or firefly luciferase-O-phosphate (bio- Reprobing
luminescence) are used for AP systems (Olesen et al., 2000),
whereas HRP systems commonly use Luminol/H2O2/p- Multiple replicas can be made in the same blotting session
iodophenol (Durrant, 1990). These reagents are all avail- by replacing the blotting membrane at intervals during the
able as commercial kits. Some brands of nitrocellulose, transfer step. However, the blots will inevitably vary in
however, contain impurities that may interfere with firefly protein content. It is also possible to reuse a developed
luciferase. Instead of photographic films the use of photon- immunoblot or probe the same blot with sets of differently
counting camera systems may increase sensitivity in chemi- visualized antibodies (Kaufmann et al., 2000). Lumines-
luminescence further. Owing to their high sensitivity, cent blots are easier to reprobe because the enzyme reaction
chemiluminescence detection methods work with more di- products do not precipitate on the membrane and two film
lute antibody and secondary reagent solutions. See also: images may be overlayed (Patel et al., 1997). A covalent
Luciferases and Light-emitting Accessory Proteins: Struc- fixation step using glutaraldehyde, divinyl sulfone or other
tural Biology crosslinking agents may make it possible to use a reacted
In multiplexing approaches using di- or trichromatic blotting membrane for a number of reprobings, but may
detection systems (Top et al., 2001; Martin et al., 2003) on lead to loss of epitopes, i.e. less antibody reactivity. Alter-
PVDF membranes a fluorescently labelled amine-reactive natively, membranes that bind proteins covalently may
chemical is combined with a fluorogenic enzyme substrate be used for such applications (Bjerrum et al., 1987;
activated by enzyme-labelled immunoreagents (dichro- Pemawansa et al., 1990). Reprobing cannot be generally
matic system). Addition of a fluorochrome-conjugated recommended.

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 Western Blotting: Immunoblotting

Other probes interfering impurities in sample buffers and gels; (2) pri-
mary antibody, to control for the presence of immuno-
The focus of this chapter is on immunodetection on blots. globulin in the antigen preparation and/or direct binding of
However, numerous other types of probes may be utilized the second antibody to components in the antigen prepa-
on transferred and immobilized protein separation pat- ration; (3) secondary antibody, to control for enzyme
terns to identify, e.g. glycoproteins, enzymes, receptors and activities in the antigen preparation and (4) the linking an-
ligands. Some of these approaches have earned their own tibodies (in multilayer techniques), to control for cross-
names, such as far-western (or ligand) blotting which is reactions between antigens on blots and second or third
used for applications where protein–protein interactions layer antibodies or nonimmunological interactions
are identified and where none of the proteins is an antibody. between the detecting system and primary antibodies.
The subsequent detection systems usually then depend on The nature of the detecting antibodies (xeno-, allo- or
antibody detection. Sometimes, the blotted protein located auto-antibodies) also implicates different controls
on the membrane is called the prey protein and the protein (Bjerrum et al., 1988). The emergence on blotted proteins
probes the bait protein (Edmondson and Roth, 2001; of new epitopes that are not present in the native molecule
Schechtman et al., 2003; Hall, 2004; Wu et al., 2007). In (neoepitopes) is also a specificity issue depending on the
contrast to immunoblotting which benefits from the uni- applications, but problems with low-affinity interactions
formity of antibody reagents these important methods re- and loss of reactivity with denatured proteins (cf. below)
quire optimization on a case-by-case basis. Additionally, as are much more prevalent. See also: Affinity of Antigen–
in any blotting method, the proper negative controls are Antibody Interactions
crucial (Howell et al., 2006).

Monoclonal versus polyclonal antibodies

Specificity Problems The use of monoclonal antibodies instead of monospecific
polyclonal antibodies often alleviates problems connected
The outcome of immunoblotting experiments is very de- with specificity. A prerequisite is that the monoclonal an-
pendent on the proper technical handling of the different tibody reacts with the immobilized antigen, which is prone
steps and on the purity and specificity of the antibodies to be more or less denatured after being subjected to SDS,
employed. Whereas artefacts and technical optimization reducing agents, methanol and heat during the procedure.
have been systematically described (Bjerrum et al., 1988), A dot immunobinding screening procedure with SDS-de-
the problems related to specificity probably need more at- natured antigen will be helpful in deciding if reactivity can
tention. The problems are typically caused by the high be expected. In some cases less than 1/5 of the monoclonals
sensitivity of immunoblotting, which visualizes antigen – reacting with native proteins also react with the SDS-de-
antibody reactions – including those of low affinity and natured protein (Bjerrum et al., 1987). However, if only
therefore of less specificity – that are not detected by other monoclonals reacting with native epitopes are available,
immunochemical methods. It is not possible to identify then blotting experiments still can be conducted, e.g. by
nonspecific bands simply by the way they look or by their using conditions that are as mild as possible during
staining intensity. A checkerboard titration with serial (1) sample preparation (no boiling, no reducing agents);
dilutions of both antigen and antibody can be used to (2) separation (isoelectric focusing or gel electrophoresis
establish an optimal signal-to-noise ratio and to suppress using native gels) and (3) transfer with no SDS and reduced
low-specificity bands. Further, incubation and washing methanol present. See also: Monoclonal Antibodies
conditions and times can be adjusted. Polyclonal antibodies almost always contain specificities
that react with conformation-insensitive epitopes in SDS-
Importance of controls denatured proteins. The problem with polyclonal antibod-
ies is that even in the so-called monospecific preparations
Relevant detection controls are indispensable in all there is often additional reactivity revealed in blotting.
immunoblotting experiments (Bjerrum et al., 1988). As is To get immunoblotting-grade monospecificity, unwanted
the case in immunohistochemistry, the first level or ab- antibody reactivity that is due to impurities in the im-
sorption controls validate the specificity of the primary munogen or to the presence of reactive background im-
antigen–antibody interaction. Solid-phase absorption munoglobulins must be removed using solid-phase
controls are preferred. To control for cross-reactivity with adsorption procedures (Bjerrum et al., 1988).
natural antibodies preimmune (nonimmune) or hyperim-
mune serum (against unrelated antigen) should be used as
controls in parallel with the primary antibody.
Second level or staining controls evaluate other factors Applications
involved in unwanted staining, such as the performance of
the detection system and of the antigen preparation. These Immunoblotting methods are in effect enzyme-linked
controls involve the omission of (1) antigen, by blotting of a immunosorbent assays (ELISAs) where the antigens are
blank gel or of an irrelevant antigen to control for immobilized in a pattern defined by molecular properties

ENCYCLOPEDIA OF LIFE SCIENCES # 2009, John Wiley & Sons, Ltd. www.els.net 5
 Western Blotting: Immunoblotting

(e.g. molecular size). The evolution of specificity and titres Limitations

in experimentally induced antibodies (immunization
protocols) can be very conveniently followed by blotting Most limitations of the blotting techniques are associated
of separated antigen mixtures. This is of paramount im- with the interpretation of the significance of a stained band,
portance for demonstrating the monospecificity of i.e. with the balance between sensitivity and specificity (as
antibodies used for immunoaffinity chromatography, discussed above). Mobilization of the molecules in the
immunohistochemistry and for ELISAs (Choi et al., transfer step, the preservation of the separation pattern
2005). In dot, slot or line immunoblotting purified anti- during the transfer, the capacity and strength of analyte
gens are deposited in arrays or as lines on membranes and binding to the blotting membrane, loss of analyte reactivity
thus no separation is integrated into the blotting process. and sensitivity and linearity of detection reagent signals are
These approaches constitute a convenient way to present a all factors that may impede quantitation. It is clear that
number of designated antigens in a predetermined pattern harsh methods necessary to obtain good separations or
for testing for reaction with antibodies, e.g. in the routine good transfers may be incompatible with subsequent de-
testing of patient sera for auto-antibodies in chronic tection steps that depend on molecular recognition of de-
immunoinflammatory conditions (González-Buitrago, fined three-dimensional structures and that separation in
2006) or as a convenient way of screening for antibody the presence of interfering substances may influence bind-
specificities and antibody reactivity patterns in diseased ing of proteins to the membrane as well as the detection by
and control individuals (Qiu et al., 2004). When a pure general and specific reagents.
immunogen is not available the technique is also very useful By virtue of being a multistep procedure, blotting is also
for picking out clones of monoclonal antibodies reacting limited with regard to the possibilities for automation and
with bands of interest in immunoblotting-based screening standardization. It is difficult to perform blotting in mi-
(Singer et al., 2008). See also: Affinity Chromatography; croformats because of the need for a sufficient resolution in
Enzyme-linked Immunosorbent Assay; Immunohisto- the separation step. Prefabricated strips with immobilized
chemical Detection of Tissue and Cellular Antigens separated proteins are available for human immunodefi-
In microbiology, immunoblotting has been applied on a ciency virus (HIV) antibody profiling. However, with the
very wide range of organisms (Vianna et al., 2006; de exception of line blots of purified antigens, application ar-
Lemos et al., 2007). In clinical chemistry immunoblotting eas where ready-made blots for antibody detection can be
methods are routinely applied for the serodiagnosis and offered are at present few and quantitative ELISAs are
monitoring of infectious, autoimmune (González- preferable when a single or a few important antigens are
Buitrago, 2006; Tamby et al., 2007) allergic diseases involved.
(Lewis et al., 2005; Weghofer et al., 2005) and degenera-
tive diseases (Singer et al., 2008). For example, immuno-
blotting has allowed elucidation of the fine specificities of
autoimmune antinuclear antibodies that were earlier clas- References
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