![]() ![]() Repeatability of Piezo-Electric Pipetting Sets Upper Bounds for Performance We provide an initial set of data that investigates such validity across a set of evaluated antibodies which will continue to grow and be publicly available. We show that results from microwestern scale to regular western. One major thrust of LINCS is improving data FAIR-ness (Findable, Accessible, Interoperable, Reusable) 28, and this particular application of microwestern is one aspect of LINCS focusing on reagent validation that is critical in such endeavors. ![]() We have implemented the microwestern array in the context of our NIH Library of Integrated Network Cellular Signatures (LINCS) Data Generation Center 25, 26, 27. The major difference is that lysates are spotted onto the surface of a gel via piezo-electric pipetting, which allows for incubation with up to 192 antibodies (96 × 2 colors) via a gasketed hybridization plate after transfer to the membrane. The microwestern process is very similar to the regular “macro” western lysates are in an SDS-containing buffer and proteins are separated by molecular weight via electrophoresis, transferred to a membrane, and incubated with antibodies for detection (Fig. The purpose of this paper is not to report on further development of the microwestern, but rather to show a potential application of the microwestern for this important quantitative validation task. The microwestern array was originally developed in 2011 in the Jones lab at the University of Chicago 23, 24. This of course considers that other important aspects of antibody validation, such as specificity via genetic approaches, are already validated 11. Here, we focus on showing how a meso-scale western blotting platform called microwestern array can help provide information to assess the validity of quantitative data from western blots-a form of antibody validation. In fact, RPPA protocols report using western blotting as the method for validating antibodies for RPPA use 22. Reverse phase protein array (RPPA) is a well-established method for quantitative data from cell and tissue lysates 19, 20, 21, 22, but it does not separate proteins by molecular weight, and therefore has more stringent requirements for antibody validity. While the western blot is often considered semi-quantitative or qualitative, it can be quantitative with infrared fluorescence-based detection 13, 14, 15, 16, 17, 18. One major application of antibodies in both large and small labs is the western blot. ![]() Antibody validity is highly dependent on biological context and the assay itself 10, 11, 12. For example, qualitative inference from immunohistochemistry may be possible, but acquiring quantitative data from flow cytometry may not be with the same antibody and cell system. The intended application for the antibody brings potentially different criteria and stringency for their use. Antibodies are widely-used critical tools in a variety of biomedical research assays, but they are not always suitable for the application of interest. While there are many potential reasons for lack of reproducibility, one major reason relates to research reagents, including antibodies 10, 11, 12. Scientific research, and in particular that in the biomedical field, has come under harsh scrutiny and debate of late due to questions of reproducibility 1, 2, 3, 4, 5, 6, 7, 8, 9. Importantly, we demonstrate that results from microwestern analyses scale to normal “macro” western for a subset of antibodies. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with 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). Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. ![]()
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