{"id":4198,"date":"2023-12-07T10:36:30","date_gmt":"2023-12-07T10:36:30","guid":{"rendered":"https:\/\/zobio.com\/?p=4198"},"modified":"2023-12-11T08:38:37","modified_gmt":"2023-12-11T08:38:37","slug":"positive-impact-of-dragonfly-in-biochemical-assay","status":"publish","type":"post","link":"https:\/\/zobio.com\/online\/blog\/positive-impact-of-dragonfly-in-biochemical-assay\/","title":{"rendered":"Positive Impact of Dragonfly\u2122 Discovery in Biochemical Assay"},"content":{"rendered":"<div id=\"pl-4198\"  class=\"panel-layout\" ><div id=\"pg-4198-0\"  class=\"panel-grid panel-has-style\" ><div class=\"panel-row-style panel-row-style-for-4198-0\" ><div id=\"pgc-4198-0-0\"  class=\"panel-grid-cell\" ><div id=\"panel-4198-0-0-0\" class=\"so-panel widget widget_sow-editor panel-first-child panel-last-child\" data-index=\"0\" ><div class=\"with-icon panel-widget-style panel-widget-style-for-4198-0-0-0\" ><div\n\t\t\t\n\t\t\tclass=\"so-widget-sow-editor so-widget-sow-editor-base\"\n\t\t\t\n\t\t><h2 class=\"widget-title\">Positive Impact of Dragonfly\u2122 Discovery in Biochemical Assay<\/h2>\n<div class=\"siteorigin-widget-tinymce textwidget\">\n\t<p>Biochemical assays are commonly used to assess how organic compounds impact the activity of a protein of interest (POI). ZoBio frequently employs biochemical assays to validate compounds that have been identified as interacting with the POI through orthogonal biophysical assays like <strong>S<\/strong>urface <strong>P<\/strong>lasmon <strong>R<\/strong>esonance (SPR) and <strong>N<\/strong>uclear <strong>M<\/strong>agnetic <strong>R<\/strong>esonance (NMR). To enhance the efficiency and reliability of the biochemical assay workflow, ZoBio has purchased a Dragonfly\u2122 Discovery, a valuable tool known for its ability to significantly improve assay performance by facilitating high-throughput, accurate, and programmable liquid dispensing. In the example below, we demonstrate how the Dragonfly\u2122 Discovery allowed us to execute a challenging assay with accuracy in a high throughput manner. Furthermore, the capability to dispense multiple reagents, makes the instrument suitable for complex studies of the <strong>M<\/strong>ode <strong>o<\/strong>f <strong>A<\/strong>ction of inhibitors.<\/p>\n<p>The ADP-Glo\u2122 lipid kinase assay is a biochemical assay designed to detect the phosphorylation of a lipid substrate by lipid kinases. We used this kit to characterize the IC<sub>50<\/sub> of inhibitors for a class I lipid kinase,\u00a0 PI3K\u03b1 which phosphorylates PIP<sub>2<\/sub> to generate PIP<sub>3<\/sub>. In order to be cost-effective, the kinase reaction had to be performed in a small, 6 \u00b5l volume in a 384-wellplate. Each assay component including the compound to be tested, PI3K\u03b1, ATP and the PIP2 substrate needed to be dispensed accurately into the wells in volumes ranging from 1 to 2 \u00b5l to initiate the kinase reaction. After an incubation period, the reaction was quenched and eventually the accumulated ADP was converted into luminescence light by addition of ADP-Glo detection solutions. Our existing 384-channel pipetting robots faced limitations in handling small volumes accurately. The result was a need for manual dispensing and mixing of components, which significantly impacted the efficiency and reliability of the assay. Our researchers could process a maximum of only 15 compounds per day, making the process time-consuming and inefficient for data collection. The manual pipetting, especially when dealing with the viscous PIP2 substrate solution, led to poor data reproducibility. This inconsistency in the pipetting process introduced errors and variability in the assay results.<\/p>\n<p>To address these challenges and improve the assay workflow, ZoBio scientists integrated the Dragonfly\u2122 Discovery into their processes. This sample handling automation resulted in a several significant improvements. \u00a0The scientists were capable of dispensing reagents at high speed, taking less than 1 minute to process a 384-well plate while significantly reducing variation between wells. This speed and accuracy even extended to the viscous lipid containing solutions needed for PI3ka activity. The assay throughput was improved significantly without compromising the assay performance as indicated by a Z' value greater than 0.8, which is a common measure of assay quality, indicating the suitability of the assay for high-throughput screening (<strong>Figure 1<\/strong>). The introduction of the Dragonfly\u2122 Discovery significantly enhanced the throughput of the assay, while at the same time reducing the need for replicate measurements. This not only saved time but also minimized reagent consumption and lowered the overall cost per assay point.<\/p>\n<p><img decoding=\"async\" class=\"wp-image-4211 aligncenter\" src=\"https:\/\/zobio.com\/online\/online\/wp-content\/uploads\/2023\/12\/Figure-1_web-3-300x157.png\" alt=\"\" width=\"1018\" height=\"533\" srcset=\"https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-1_web-3-300x157.png 300w, https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-1_web-3-1024x535.png 1024w, https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-1_web-3-768x401.png 768w, https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-1_web-3.png 1200w\" sizes=\"(max-width: 1018px) 100vw, 1018px\" \/><\/p>\n<p><em><strong>Figure 1<\/strong>. Lipid kinase ADP-Glo workflow <strong>A<\/strong>)\u00a0samples dispensed by handheld electronic pipettes and <strong>B<\/strong>)\u00a0samples\u00a0 dispensed by Dragonfly\u2122 Discovery.<\/em><\/p>\n<p>Our Dragonfly\u2019s flexibility allows us to prepare complicated assays since it can pipette up to 10 different components. Since we sought allosteric inhibitors of PI3K\u03b1, it was important to validate this <strong>M<\/strong>ode <strong>o<\/strong>f <strong>A<\/strong>ction for biophysically selected hits using an enzyme assay that monitors the IC50 of an inhibitor while increasing the concentration of ATP in the lipid kinase assay (<strong>Figure 2<\/strong>). Various concentrations of ATP were prepared by Dragonfly while keeping the compound concentration range constant. Idelalisib, a well characterized ATP site inhibitor, bound apo-PI3K\u03b1 with a K<sub>D<\/sub> of 0.7 \u00b5M in SPR. The IC<sub>50<\/sub> of Idelalisib shifted from 0.4 to 25 \u00b5M as the concentration of ATP increased from 3 to 625 \u00b5M, confirming its ATP competitive mechanism. In contrast, the IC<sub>50<\/sub> of Inhibitor X did not significantly change as the ATP concentration increased, suggesting an allosteric mechanism. This mechanism of compound X was orthogonally confirmed using \u00a0SPR where binding was shown to be non-competitive with Idelalilsib.<\/p>\n<p><img decoding=\"async\" class=\"alignnone wp-image-4200 aligncenter\" src=\"https:\/\/zobio.com\/online\/online\/wp-content\/uploads\/2023\/12\/Figure-2_web-300x157.png\" alt=\"\" width=\"890\" height=\"466\" srcset=\"https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-2_web-300x157.png 300w, https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-2_web-1024x535.png 1024w, https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-2_web-768x401.png 768w, https:\/\/zobio.com\/online\/wp-content\/uploads\/2023\/12\/Figure-2_web.png 1200w\" sizes=\"(max-width: 890px) 100vw, 890px\" \/><\/p>\n<p><em><strong>Figure 2<\/strong>. Determination of the mode of action of PI3K\u03b1 inhibitors.<\/em><\/p>\n<p>Stay tuned for insights, discoveries, and a closer look at the transformative role of technology in shaping our research endeavors.<\/p>\n<p>&nbsp;<\/p>\n<\/div>\n<\/div><\/div><\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"<p>Biochemical assays are commonly used to assess how organic compounds impact the activity of a protein of interest (POI). ZoBio frequently employs biochemical assays to validate compounds that have been identified as interacting with the POI through orthogonal biophysical assays like Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR). To enhance the efficiency and reliability of the biochemical assay workflow, ZoBio has purchased a Dragonfly\u2122 Discovery, a valuable tool known for its ability to significantly improve assay performance by facilitating high-throughput&#8230;<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"gpsa_enabled":false,"gpsa_required_form_ids":[],"gpsa_require_unique_form_submission":false,"gpsa_access_behavior":"show_message","gpsa_form_redirect_path":"","gpsa_requires_access_message":"","gpsa_access":[],"gpsa_content_loading_message":"","footnotes":""},"categories":[13],"tags":[],"class_list":["post-4198","post","type-post","status-publish","format-standard","hentry","category-blog"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.1.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Positive Impact of Dragonfly\u2122 Discovery in Biochemical Assay - ZoBio - Drug Discovery Technology<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/zobio.com\/online\/blog\/positive-impact-of-dragonfly-in-biochemical-assay\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Positive Impact of Dragonfly\u2122 Discovery in Biochemical Assay - ZoBio - Drug Discovery Technology\" \/>\n<meta property=\"og:description\" content=\"Biochemical assays are commonly used to assess how organic compounds impact the activity of a protein of interest (POI). 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