A duplex approach for immunochemical staining and typing of proteins in Western blots

Kuczius, Thorsten; Brandstädter, Lars; Karch, Helge; Langeveld, J.P.M.

doi:10.1016/j.ab.2010.10.018

Cited by 2 publications

(1 citation statement)

References 35 publications

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“…Many attempts have been made to improve the method throughput and detection such as multistrip Western blotting , a duplex approach for immunochemical staining , semidry transfer iBlot system , detection sensitivity using dual wavelength fluorescence , and quantum dot fluorescence . In general, the reproducibility of manual Westerns is poor because of the SDS‐PAGE gel heterogeneity, the difficulty of controlling protein transfer from the gel to the membrane, as well as immunostaining and detection methods . Even with these pitfalls many authors have attempted to perform quantitative Western blots.…”

Section: Introductionmentioning

confidence: 99%

Qualitative and quantitative evaluation of Simon™, a new CE‐based automated Western blot system as applied to vaccine development

Rustandi

Loughney²,

Hamm³

et al. 2012

Electrophoresis

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Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.

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Section: Introductionmentioning

confidence: 99%

Qualitative and quantitative evaluation of Simon™, a new CE‐based automated Western blot system as applied to vaccine development

Rustandi

Loughney²,

Hamm³

et al. 2012

Electrophoresis

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Nonstripping “Rainbow” and Multiple Antigen Detection (MAD) Western Blotting

Krajewski

Tsukamoto

Huang

et al. 2015

Methods in Molecular Biology

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A variation of immunoblotting method (the "Rainbow Western"), permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping off prior antibodies. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple reprobings of the same membrane with different primary antibodies (≥12), retaining strong signal intensities for all sequential antibody probings. The procedure utilizes horseradish peroxidase (HRPase)-based detection with both a chemiluminescent and colorimetric substrate. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. In the "Rainbow Western," four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same membrane. By allowing large amounts of data to be obtained from a single blot, the MAD-immunoblotting and Rainbow Western methods have the potential for researchers to compare the expression of several proteins within a single biological sample. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples is minute or only available on a one-time basis.

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A duplex approach for immunochemical staining and typing of proteins in Western blots

Cited by 2 publications

References 35 publications

Qualitative and quantitative evaluation of Simon™, a new CE‐based automated Western blot system as applied to vaccine development

Qualitative and quantitative evaluation of Simon™, a new CE‐based automated Western blot system as applied to vaccine development

Nonstripping “Rainbow” and Multiple Antigen Detection (MAD) Western Blotting

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