Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot.

The article presents a new method of immunoblotting for simple, rapid, and highly sensitive detection of proteins. Electrophoretic separation of sample is carried out under non-denaturing conditions in a thin conductive layer between cellulose membranes without polyacrylamide gel. The membrane surface is preliminarily modified with azidophenyl groups to photochemically immobilize proteins in situ. For visualization of protein bands, the membranes are treated with magnetic beads coated with specific antibodies, unbound particles are then removed with a magnet. The detection limit in the model system with biotinylated BSA and magnetic beads coated with streptavidin reaches 10 fg or about 105 molecules, while the total blotting time does not exceed 5 min. The method was applied for detection of IgA in a sample of human exhaled air. The method can be used for the analysis of various complex biological samples containing low amounts of the analyte.

Fabrication of an Open Microfluidic Device for Immunoblotting.

Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an "enclosed" microchannel for PAGE and blotting to an "open" PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.

Use of IgY antibody to recombinant avian reovirus σC protein in the virus diagnostics.

Avian reovirus (ARV) is an important agent of several diseases causing considerable losses in poultry farming. An outer capsid protein (σC) of ARV, is known as a virus-cell attachment protein essential for virus infectivity. In this study, the σC gene of ARV was cloned and expressed in Escherichia coli. The expressed recombinant protein was used as immunogen for raising a specific IgY antibody in laying hens. At 14 weeks post immunization, the antibody titers in serum and egg yolk reached 302,000 and 355,000, respectively. The IgY antibody was capable to neutralize ARV in BHK-21 cells and it strongly reacted in ELISA with ARV but not with heterologous viruses. The IgY antibody detected ARV in field samples of infected animal tissues in dot blot assay. These results suggest that an efficient, economic and rapid diagnostics of ARV can be performed routinely using the IgY antibody against a recombinant ARV σC protein.

Comparison of Stain-Free gels with traditional immunoblot loading control methodology.

Loading controls are necessary for semiquantitative Western blotting to compensate for loading errors. Loading control methods include the reprobing of membranes with an antibody against a constitutively expressed protein or staining the membrane with a total protein stain. We compared the loading control performance of recently released Stain-Free (SF) gels with Sypro Ruby (SR) and reprobing using ß-actin. SF gels demonstrated superior performance in that they were faster, required fewer steps and consumables, and allowed the quality of electrophoresis and Western transfer to be assessed before committing to costly and time-consuming Western blots.

Field monitoring of plant-growth-promoting rhizobacteria by colony immunoblotting.

Inoculant plant-growth-promoting bacteria are emerging as an important component of sustainable agriculture. There is a need to develop inexpensive methods for enumerating these organisms after their application in the field, to better understand their survival and impacts on yields. Immunoblotting is one potential method to measure viable cells, but the high cost of the conventionally used nylon membranes makes this method prohibitive. In this study, less expensive alternative materials such as filter papers, glossy photo papers, and transparencies for the purpose of colony immunoblotting were evaluated and the best substance was chosen for further studies. Whatman filter paper No. 541 combined with a 0.01 mol·L(-1) H(2)SO(4) rinsing step gave similar results to nylon membranes but <20% of the overall cost of the original colony immunoblotting assay. The application of the modified immunoblot method was tested on nonsterile clay soil samples that were spiked with high numbers (>10(7) CFU·g(-1)) of the plant-growth-promoting bacteria Pseudomonas fluorescens , Azospirillum brasilense , or Rhizobium leguminosarum . The modified protocol allowed the identification and recovery of over 50% of the inoculated cells of all three strains, amidst a background of the native soil microflora. Subsequently, the survival of P. fluorescens was successfully monitored for several months after application to field-grown rice at Jerilderie, New South Wales, Australia, thus validating the procedure.

Polyacrylamide gel photopatterning enables automated protein immunoblotting in a two-dimensional microdevice.

We demonstrate a two-dimensional microfluidic architecture that integrates polyacrylamide gel electrophoresis (PAGE) with immunoblotting in a fully automated format. This assay is designed to overcome performance limitations of conventional slab-gel immunoblotting, including multiple manual interventions, low-throughput transfer and blotting, and substantial consumption of reagents and sample. To unify PAGE with blotting in one device, this microfluidic approach makes use of high-resolution regional photopatterning of multiple polyacrylamide gel elements, and automated electrophoretic transport. A complete native immunoblot of free prostate specific antigen from human seminal fluid is demonstrated in less than 5 min. Further, the characterization of post-PAGE electrophoretic transfer showed high efficiency and minimal sample dispersion.

An inexpensive technique for dot blotting in a 96-well format.

Dot blotting for the quantification of proteins usually requires expensive devices. Here we present an inexpensive way to facilitate dot blotting in a 96-well format. An agarose gel was used as sample reservoir, and proteins were electrophoretically transferred from the gel to a membrane. Dots produced by this technique were applied to reproducible immunoquantification of proteins such as beta-actin and doublecortin.

Probe-target hybridization depends on spatial uniformity of initial concentration condition across large-format chips.

Diverse assays spanning from immunohistochemistry (IHC), to microarrays (protein, DNA), to high-throughput screens rely on probe-target hybridization to detect analytes. These large-format 'chips' array numerous hybridization sites across centimeter-scale areas. However, the reactions are prone to intra-assay spatial variation in hybridization efficiency. The mechanism of spatial bias in hybridization efficiency is poorly understood, particularly in IHC and in-gel immunoassays, where immobilized targets are heterogeneously distributed throughout a tissue or hydrogel network. In these systems, antibody probe hybridization to a target protein antigen depends on the interplay of dilution, thermodynamic partitioning, diffusion, and reaction. Here, we investigate parameters governing antibody probe transport and reaction (i.e., immunoprobing) in a large-format hydrogel immunoassay. Using transport and bimolecular binding theory, we identify a regime in which immunoprobing efficiency (η) is sensitive to the local concentration of applied antibody probe solution, despite the antibody probe being in excess compared to antigen. Sandwiching antibody probe solution against the hydrogel surface yields spatially nonuniform dilution. Using photopatterned fluorescent protein targets and a single-cell immunoassay, we identify regimes in which nonuniformly distributed antibody probe solution causes intra-assay variation in background and η. Understanding the physicochemical factors affecting probe-target hybridization reduces technical variation in large-format chips, improving measurement precision.

Double-blotting: a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures.

Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption but they may work in specific applications and be ineffective in other ones. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.

Grid-immunoblotting.

Grid-Immunoblotting is a fast, simple, and efficient method for simultaneously testing multiple allergens utilizing small amount of antibody.

Slice blotting.

Slice blotting is a technique for recording the spatial distribution of extracellular signaling molecules released from thin slices of living tissue. Slices are positioned on the surface of a membrane that can trap secreted substances diffusing from the tissue. The pattern of membrane-bound antigens is subsequently visualized by immunoblotting.

Localizing proteins by tissue printing.

The simple technique of making tissue prints on appropriate substrate material has made possible the easy localization of proteins, nucleic acids, carbohydrates, and small molecules in a tissue-specific mode. Plant tissues can be used to produce prints revealing a remarkable amount of anatomical detail, even without staining, which might be used to record developmental changes over time. In this chapter we will focus on the protocols for the localization of proteins and glycans using antibodies or lectins, probably the most frequently used application, but the localization of other molecules is reported and the sources indicated.

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

In the functional proteome era, the proteomic profiling of clinicopathologic annotated tissues is an essential step for mining and evaluations of candidate biomarkers for disease. Previously, application of routine proteomic methodologies to clinical tissue specimens has provided unsatisfactory results. Multiplex tissue immunoblotting is a method of transferring proteins from a formalin-fixed, paraffin-embedded tissue section to a stack of membranes which can be applied to a conventional immunoblotting method. A single tissue section can be transferred to up to ten membranes, each of which is probed with antibodies and detected with fluorescent tags. By this approach, total protein and target signals can be simultaneously determined on each membrane; hence each antibody is internally normalized. Phosphorylation-specific antibodies as well as antibodies that do not readily work well with paraffin-embedded tissue are applicable to the membranes, expanding the menu of antibodies that can be utilized with formalin-fixed tissue. This novel platform can provide quantitative detection retaining histomorphologic detail in clinical samples and has great potential to facilitate discovery and development of new diagnostic assays and therapeutic agents.

Sequential use of immunoblots for characterization of autoantibody specificities.

Sera of patients with systemic autoimmune diseases frequently contain autoantibodies to nuclear autoantigens. Immunoblotting of recombinant and native autoantigens is a commonly used technique for the identification and characterization of autoantibody specificities. Here, we describe an easy procedure that facilitates the comparison of antibody specificities by reusing the same immunoblot at least three times in order to detect an abundantly expressed autoantigen in total cellular extracts.

Affinity immunoblotting for analysis of antibody clonotype distribution in a lupus patient developing anti-Ro 60 over time.

We describe a sensitive and specific method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera collected over several years were separated by flatbed isoelectric focusing and analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane. When the Ro 60-coated nitrocellulose was laid over the surface of the IEF gel, the antibodies present on the surface of the acrylamide gel bound the Ro antigen on the nitrocellulose. Tween-20 was used to prevent nonspecific binding. The bound IgG clonotypes were detected using alkaline phosphatase conjugated anti-IgG. The patient's sera demonstrated an oligoclonal response to the Ro 60 autoantigen that increased in complexity and affinity over time.

Cationic electrophoresis and eastern blotting.

Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases, electrophoresis in the presence of positively charged detergents like cetyltrimethylammonium bromide may work considerably better. Methods for electrophoresis, staining, and blotting of such gels are presented.

A miniaturized blotting system for simultaneous detecting of different autoantibodies.

Sera of tumor patients frequently contain autoantibodies to tumor-associated antigens. Here we describe a miniaturized immunoblot platform allowing us to screen sera of patients for the presence of autoantibodies to ten autoantigens in parallel.

Whole and strip nitrocellulose membrane as well as a new-line-immunoblotting of antigen using the chemiluminescence technique.

There are a number of techniques in the scientific world that researchers use to detect specific antigens. One such technique that has provided many advantages over typical immunochemical staining is chemiluminescence. The emission of visible radiation by compounds once exposed to sunlight has been known for centuries and currently is the main principle for chemiluminescence. Here, we introduce three different chemiluminescence techniques that are widely used in immunodetection of antigens: (a) whole membrane chemiluminescence detection, (b) strip membrane chemiluminescence detection, and (c) new line blotting chemiluminescence.

A fluorescent codetection system for immunoblotting and proteomics through ECL-Plex and CyDye labeling.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

Simultaneous immunoblotting analysis with activity gel electrophoresis and 2-D gel electrophoresis.

Diffusion blotting method can couple immunoblotting analysis with another biochemical technique in a single polyacrylamide gel, however, with lower transfer efficiency as compared with the conventional electroblotting method. Thus, with diffusion blotting, a protein blot can be obtained from a sodium dodecyl sulfate polyacrylamide gel for zymography assay, from a native polyacrylamide gel for electrophoretic mobility shift assay (EMSA), or from a two-dimensional (2-D) polyacrylamide gel for large-scale screening and identification of a protein marker. Therefore, a particular signal in zymography, EMSA, and 2-D gel can be confirmed or identified by simultaneous immunoblotting analysis with a corresponding antiserum. These advantages make diffusion blotting desirable when partial loss of transfer efficiency can be tolerated or can be compensated by a more sensitive immunodetection reaction using enhanced chemiluminescence substrates.