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• W2779876871 abstract "ABSTRACT Western blotting is often considered a semi-quantitative or even qualitative assay for assessing changes in protein or protein post-translational modification levels. Fluorescence-based measurement enables acquisition of quantitative data in principal, but requires determining the linear range of detection for each antibody—a labor-intensive task. Here, we describe the use of a high-throughput western blotting technique called microwestern array to more rapidly evaluate suitable conditions for quantitative western blotting with particular antibodies. We can evaluate up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a surprisingly high proportion of investigated antibodies (17/22) are suitable for quantitative use, and that lack of validity might often be a consequence of lysate composition rather than antibody quality. Linear range for all validated antibodies is at least 8-fold, and in some cases nearly two orders of magnitude. That range could be greater as the presented tests did not find a limit for many antibodies. We find that phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection, but total antibodies generally required higher working concentrations, suggesting phospho-specific antibodies may be generally higher affinity. Importantly, we demonstrate that results from microwestern analyses scale to normal “macro” western for a subset of antibodies. These data indicate that with initial validation, many antibodies can be readily used quantitatively in a reproducible manner. Antibody validation data and standard operating procedures are available online ( www.birtwistlelab.com/protocols and www.dtoxs.org )." @default.
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• W2779876871 date "2017-11-17" @default.
• W2779876871 modified "2023-09-27" @default.
• W2779876871 title "Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array" @default.
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• W2779876871 doi "https://doi.org/10.1101/221523" @default.
• W2779876871 hasPublicationYear "2017" @default.
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