Simple Western: Bringing the Western Blot into the Twenty-First Century.

The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.

Translating Current Bioanalytical Techniques for Studying Corona Activity.

The recent discovery of the biological corona is revolutionising our understanding of the in vivo behaviour of nanomaterials. Accurate analysis of corona bioactivity is essential for predicting the fate of nanomaterials and thereby improving nanomedicine design. Nevertheless, current biotechniques for protein analysis are not readily adaptable for analysing corona proteins, given that their conformation, activity, and interaction may largely differ from those of the native proteins. Here, we introduce and propose tailor-made modifications to five types of mainstream bioanalytical methodologies. We specifically illustrate how these modifications can translate existing techniques for protein analysis into competent tools for dissecting the composition, bioactivity, and interaction (with both nanomaterials and the tissue) of corona formed on specific nanomaterial surfaces.

The fastest Western in town: a contemporary twist on the classic Western blot analysis.

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

Investigating protein-protein interactions by far-Westerns.

The identification of protein interaction partners can often elucidate the function of the protein under investigation based on the "guilty by association" concept. Furthermore, the binding event between two proteins can be used as a functional assay when no such assay is available. Despite the large number of advanced techniques that are currently available for studying protein-protein interactions, far-Westerns or blot overlays are still very commonly used in the average laboratory setting due to their powerfulness. This is due to the simplicity and clarity in the results that they produce. Here, the details and mechanics of far-Westerns are discussed to help the reader choose amongst the different variations that exist depending on the question being investigated and the materials available to them. Some examples involving unique questions are also discussed in order to educate the reader on the versatility of far-Westerns. Finally, a troubleshooting section provides the reader with an understanding of the common problems that can be encountered and how these problems can be circumvented.

Western blot profile in HIV infection.

BACKGROUND: Although the overall sensitivity and specificity of the western blot (WB) test for detection of antibodies to various viral proteins is high, there has been a substantial difference in the timing of the appearance of antibody bands and their intensities during different stages of HIV infection. AIMS: Mapping different band patterns of Western blot results and correlating them with stages of HIV infection. METHODS: We performed a retrospective study with 1,467 HIV-1 infected cases confirmed by WB test between January 2002 to July 2005, with the objective of mapping different band patterns of western blot results and determining whether the presence or absence of certain bands was associated with any specific stage of HIV infection. For the interpretation of the WB results in this study, the guidelines recommended by NACO, India were followed. RESULTS: Reactivity with all the bands was the most commonly observed WB pattern, occurring in 92.91% (1363/1467) of cases, whereas the other 7.09% showed uncommon band patterns. Of all individual bands, p31 band was the most frequently missing one, absent in 7.09% cases. On classifying the WB reactive cases by the WHO clinical staging system, 38.45% (564/1467) were in Stage 1, 47.99% (704/1467) in stages 2 and 3 and 13.56% in stage 4. Correlation of CD4 cell counts with the various uncommon band patterns showed that only 5.56% (4/72) had counts in the 200-500 cells/microl range, whereas 45.83% and 48.61% had counts of < 200 and> 500 cells/microl respectively. CONCLUSION: Interpretation of the WB band pattern in combination with clinical features may be occasionally useful in predicting the stage of HIV infection.

Detection of genetically modified organisms in foods.

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.

Human T-lymphotropic virus type I/II. Status of enzyme immunoassay and western blot testing in the United States in 1989 and 1990.

In three performance evaluation surveys, panels that consisted of human T-lymphotropic virus type I or type II (HTLV-I/II) antibody-positive and -negative plasma samples were mailed to laboratories that voluntarily participated in the Centers for Disease Control Model Performance Evaluation Program. Donor samples were identical among surveys. In each survey, more than 98% of the laboratories reported enzyme immunoassay (EIA) test results; about 11% also reported results of Western blot (WB) testing. Variation in analytic sensitivity (96.7% to 99.4%) and specificity (98.3% to 99.5%) of EIA tests was noted in the three surveys. For WB testing, no nonreactive interpretations were reported for HTLV-I/II antibody-positive samples in any survey; however, indeterminate interpretations were reported for 35.2% to 40.7% of the WB tests that were performed on HTLV-I/II antibody-positive samples. More than 95% of these indeterminate WB test interpretations were reported for HTLV-II antibody-positive samples. Although HTLV-I/II antibody tests are generally sensitive and specific, their accuracy could be further improved by increasing the specificity of EIA tests and the sensitivity of WB tests.