Masaharu Mabuchi

Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays.

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.

Multiplexed Fluorescent Immunodetection Using Low Autofluorescence Immobilon®-FL Membrane

By enabling greater signal linearity and multiplexed detection, fluorescent western immunodetection overcomes many of the inherent technical limitations associated with the traditional chemiluminescent detection method. However, the sensitivity of fluorescence detection can be severely compromised by high background autofluorescence of various blotting membranes. Here, we describe a low autofluorescence PVDF membrane (Immobilon(®)-FL membrane) optimized for fluorescent immunodetection, and we report its use in the quantitative fluorescent western immunodetection of biomarkers associated with Alzheimers disease (AD). First, membrane autofluorescence of four different commercially available blotting membranes was compared. Immobilon(®)-FL membrane exhibited the lowest autofluorescence with substantially increased detection sensitivity. We also show that the fluorescent immunodetection exhibited greatly increased linear dynamic range (two orders of magnitude, log scale) compared to the traditional chemiluminescent methods (less than one order of magnitude). Immobilon(®)-FL membrane was then used to quantify the expression levels of previously reported biomarkers associated with AD (synaptophysin, GSK3β, and GAP43). Total protein extracts from age-matched brain samples of three AD patients and three normal controls were used. Biomarker expression levels were normalized to that of a housekeeping protein (GAPDH) using multiplexed detection, conserving difficult-to-obtain biological tissue samples and minimizing experimental variation. We found that the expression of GSK3ß and GAP43 biomarkers were significantly reduced in AD brain samples compared to age-matched normal samples. In summary, combining fluorescent immunodetection with a low autofluorescent blotting membrane yields accurate and reliable multiplexed quantitation of AD biomarkers. While this report focuses on the quantitation of AD biomarkers, the described technique is applicable to comparing protein expression in other biological contexts as well.

Abstract 3483: A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples

Exosomes represent a subset of small particles secreted by many types of cells under both normal and pathological conditions. The release of microvesicles has demonstrated biological relevance; these particles act as mediators of intercellular communication both within the local microenvironment of the release site as well as systemically. Moreover, since exosomes contain RNA (messenger and miRNA) and protein (membrane-bound and cytosolic) from their cells of origin, and given that this content can be influenced by cell state, they also potentially represent a burgeoning target for biomarker discovery. Critical to understanding the physiological significance of these particles is the development of preparative techniques permitting reliable isolation of purified fractions. While numerous methods of exosome purification exist, including ultracentrifugation, immunoaffinity-based isolation using magnetic beads, precipitation, and ultrafiltration, most are plagued by sample limitations or require long and tedious workflows to achieve success. Here we present a rapid alternative method for the selective fractionation of exosomes from biological samples using an ultrafiltration device. Since the method is spin-based and dependent on size exclusion, the device has broad applications with regards to sample volume and/or type. Optimization of the protocol was aided through use of a mid infrared (MIR)-based spectroscopy platform that permits simultaneous monitoring of lysis conditions, protein quantitation, and analysis of total lipid content during exosome fractionation. Given the ultrafiltration device9s capacity for buffer exchange and sample concentration, purified fractions can be easily formatted to meet the requirements of any downstream analysis platform. To demonstrate this, resulting fractions were assayed by numerous techniques including flow cytometry, western blotting, ELISA-based assays, and electron microscopy. Citation Format: Amedeo Cappione, Sara Gutierrez, Masaharu Mabuchi, Janet Smith, Ivona Strug, Timothy Nadler. A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2014-3483

Abstract 4152: New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids.

Microvesicles, such as exosomes, are small particles secreted by many types of cells under both normal and pathological conditions such as cancer. These microparticles contain RNA and protein (both membrane-bound and cytosolic) from their cells of origin and thus represent a burgeoning resource for biomarker identification. While plasma and urine offer convenient, non-invasive access to sufficient sample volumes for screening purposes, major obstacles to this effort include the problematic issues of exosome isolation methods, the presence of over-abundant proteins or interfering cell debris, and the wide dynamic range of protein expression. It is clearly recognized in many fields that the quality of sample preparation ultimately impacts their performance in downstream analyses; this is particularly true where the target in question is a low abundant protein. Here we present an ultrafiltration-based method for the isolation of microvesicles from cell culture media as well as body fluids. To assist in process optimization, we used a novel infrared (IR) based biomolecule detection system to evaluate lysis and extraction conditions and analyze protein and lipid content. This IR system is less influenced by reducing agents and detergents than either BCA or Bradford assays. Following isolation, samples were analyzed using a rapid immunodetection technique, permitting detection of biomarkers more quickly and efficiently than ever before. Citation Format: Sara Gutierrez, Ivona Strug, Amedeo Cappione, Janet L. Smith, Masaharu Mabuchi, Timothy Nadler. New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2013-4152

Optimierung von Western Blot-Signalen mit Chemilumineszenz

ZusammenfassungWestern Blot zum Nachweis von Proteinen in Zelllysaten ist eine wertvolle Ergänzung zu immunzytochemischen Methoden. Erfolgreiche Western Blots erfordern die Optimierung mehrerer Variablen. Diese Studie zeigt die Optimierung der Chemilumineszenz-Detektion.AbstractUsing Western blotting to detect proteins in a cell lysate is a powerful, complementary approach to immunocytochemistry. Successful Western blotting is dependent on the optimization of multiple variables. In this study, we focused on optimization of chemiluminescence detection

Visualization of Unstained Protein Bands on PVDF

In 1988, two separate investigators reported a novel method of detecting unstained protein bands on polyvinylidene fluoride (PVDF) membranes using white light transillumination. This simple method exploits the intrinsic hydrophobicity of PVDF membrane, which enables the visual observation of transferred protein bands due to differential wetting patterns between protein bands and the membrane itself. This method applies only to hydrophobic PVDF membranes, because hydrophilic membranes such as nitrocellulose wet out completely, rendering the protein bands invisible by transillumination. Transilluminational protein visualization can detect submicrogram quantities of proteins while circumventing the use of protein stains, which can potentially interfere with downstream analysis such as N-terminal sequencing. In this chapter, we demonstrate efficient transilluminational protein visualization on a recently introduced low-fluorescence PVDF membrane, normally used for downstream fluorescent immunodetection.