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Western Blotting with Solutions containing Nanoliter Volumes of Antibody.

ABSTRACT: Whether screening small mammal serum during antibody production or attempting to preserve a stock of precious antibody, this protocol's western blotting method using aliquots containing nanoliter volumes of antibody will benefit researchers. Time-tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. This article provides a step-by-step protocol for detecting proteins of interest with solutions containing nanoliter volumes of antibody without altering the preceding gel electrophoresis and transfer methods. Important considerations, frequently encountered problems, and means of optimizing reproducibility are discussed. Complementary diagrams, images, and videos are provided. The protocol is demonstrated using 0.3 nanoliters of anti-serum to detect fibronectin in a human foreskin fibroblast cell line. Finally, two support protocols detailing methods of extracting proteins from cultured cells are reported. Published 2019. This article is a US Government work and is in the public domain in the USA.

SUBMITTER: Cruz Walma DA

PROVIDER: S-EPMC6727981 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Western Blotting with Solutions containing Nanoliter Volumes of Antibody.

Cruz Walma David A DA Collins Joshua W JW

Current protocols in cell biology 20190901 1

Whether screening small mammal serum during antibody production or attempting to preserve a stock of precious antibody, this protocol's western blotting method using aliquots containing nanoliter volumes of antibody will benefit researchers. Time-tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. This artic ...[more]

PMID: 31483112

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Project description:Background: The microtubule-associated protein Tau has attracted a diverse and ever-increasing research interest, with the protein being mentioned in the title/abstract of nearly 34,000 PubMed-indexed publications to-date. To facilitate studies into Tau biology and its role in neurodegeneration, a multitude of Tau-targeting antibodies have been developed, with hundreds of distinct antibody clones currently available for purchase. Nevertheless, concerns over antibody specificity and limited understanding of the performance of many of these reagents has hindered research into the protein. Methods: We have characterised the performance and specificity of 53 commercially-available Tau antibodies by Western blot, 35 of which were additionally profiled by immunohistochemistry. Antibodies were tested for their reactivity to both murine and human samples, with supporting data generated by mass spectrometry-based detection. Results: We show that presumed Tau knockout human cells continue to express residual protein, providing evidence of Tau isoforms generated by exon skipping. Our data also reveals that the binding of several antibodies presumed to detect Tau independently of any post translational modifications (i.e. “total” Tau antibodies, as well as an antibody targeting Asp421-cleaved Tau), were partially inhibited by phosphorylation. Moreover, we found that several total and phospho-Tau antibodies cross-reacted with MAP2 isoforms, while the “oligomer-specific” T22 antibody detected monomeric Tau on Western blot. With one exception, the phosphorylation-dependent Tau antibodies tested were found to not cross-react with the unphosphorylated protein. Importantly, several total and isoform-specific Tau antibodies failed to detect Tau expressed at low endogenous levels, highlighting the importance of antibody choice for studying physiological Tau expression, especially in non-neuronal cells and peripheral tissues. Conclusions: We identify Tau antibodies across all categories (total, PTM-dependent and isoform-specific) that can be employed in Western blotting and/or immunohistochemistry applications to reliably detect even low levels of Tau expression with high specificity. This work represents an extensive resource that serves as a point of reference for future studies with respect to anti-Tau antibody use and development.

Dataset Information

Western Blotting with Solutions containing Nanoliter Volumes of Antibody.

Publications

Western Blotting with Solutions containing Nanoliter Volumes of Antibody.

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