{"id":4157,"date":"2024-02-25T09:22:52","date_gmt":"2024-02-25T09:22:52","guid":{"rendered":"http:\/\/biogroupvietnam-worldpress.local\/2025\/08\/18\/total-protein-normalization-stain-free-technology-and-uvitec-imagers\/"},"modified":"2025-09-29T13:59:54","modified_gmt":"2025-09-29T06:59:54","slug":"chuan-hoa-protein-tong-so-bang-cong-nghe-stain-free-may-chup-anh-gel-uvitec","status":"publish","type":"post","link":"https:\/\/biogroupvietnam.com\/en\/new-products-technologies\/total-protein-normalization-stain-free-technology-and-uvitec-imagers\/","title":{"rendered":"Total protein normalization: Stain-Free technology and Uvitec imagers"},"content":{"rendered":"<p><strong>1. Introduction<\/strong><\/p>\n<p style=\"text-align: justify;\">In Western blotting, normalization permits to mathematically correct the levels of a target protein to an internal control, which is used as indicator of sample protein loading.<\/p>\n<p style=\"text-align: justify;\">This procedure represents a critical step and it is required to correct for loading variation, thus confirming that the observed changes represent actual differences between samples.<\/p>\n<p style=\"text-align: justify;\">Housekeeping proteins are commonly used as loading controls for Western blot normalization. However, in some experimental conditions, it is difficult to detect a good housekeeping protein, characterized by a stable expression. For this reason, normalization can be calculated on the total protein levels for each sample quantified by using TGX Stain-freeTM gels.<\/p>\n<p><strong>2. Experiment Conditions<\/strong><\/p>\n<ul>\n<li>\n<p style=\"text-align: justify;\">To detect the effect of a beta-lactam antibiotic on the expression of the glutamate transporter GLT-1 in cultured astrocytes, cells (120.000 cells\/well for 12-well plates) were treated at DIV20 with increasing doses of drug for 48 hours.<\/p>\n<\/li>\n<li>\n<p style=\"text-align: justify;\">Drug, dissolved in water, was added to the cultured medium (DMEM-HAM F10, 10%FBS, 1% Pen\/Strep).<\/p>\n<\/li>\n<li>\n<p style=\"text-align: justify;\">Astrocytes were lysed using Laemmli buffer (50 \u03bcl\/well for 12-well plates) and boiled at 95\u00b0C for 3 minutes.<\/p>\n<\/li>\n<li>\n<p style=\"text-align: justify;\">15 \u00b5l of protein lysates, corresponding to 15 \u00b5g of proteins, were loaded on 4-15% CriterionTM TGXTM Precast Midi Protein Gel (Bio-Rad) and run at 200V constant.<\/p>\n<\/li>\n<li>\n<p style=\"text-align: justify;\">After electrophoretic separation, the gel was directly placed on the UV-transilluminator (ESSENTIAL V6 System, UVITEC Ltd, UK) and activated for 60 seconds (Figure 1). Then, proteins were blotted on a nitrocellulose membrane using a semidry transfer apparatus (Trans-blot SD; Bio-Rad).<\/p>\n<\/li>\n<li>\n<p style=\"text-align: justify;\">Membranes were incubated 1 hour in blocking solution (Tris-buffered saline containing 0.1% Tween-20 [TBS-T] and 5% non-fat dry milk) and then incubated overnight (4\u00b0C) with anti-GLT1 primary antibody (Santa Cruz Biotechnology; 1:500 in 5% milk in TBS-T).<\/p>\n<\/li>\n<li>\n<p style=\"text-align: justify;\">After three washes in TBS-T, blots were incubated for 1 hour at room temperature with anti-mouse HRP-conjugated secondary antibody (1:10000 in 5% milk in TBS-T) and the immunocomplexes were visualized by using the ECL substrate kit (GenSpin, Milan, Italy) and imaged on ALLIANCE MINI HD9 system (UVITEC Ltd, UK). GLT-1 band volume measurements were performed using the UVITEC software and the value normalized to the corresponding TGX-stain lane. Background subtraction was performed by selecting \u201crolling ball\u201d for all measurements. The mean value of the untreated group (control) was set at 100% and data expressed as percentage of control.<\/p>\n<\/li>\n<\/ul>\n<p><strong>3. Conclusion<\/strong><\/p>\n<p style=\"text-align: justify;\">The selected beta-lactam antibiotic increases the expression of GLT-1 in cultured astrocytes in a dose-dependent manner (Figure 2).<\/p>\n<p style=\"text-align: center;\"><img fetchpriority=\"high\" decoding=\"async\" src=\"https:\/\/biogroupvietnam.com\/assets\/uploads\/image\/picture3.png\" alt=\"\" width=\"300\" height=\"210\" \/><\/p>\n<p style=\"text-align: center;\"><i>Figure 1. Image Of TGX Stain-Free Gel After UV Activation.<\/i><\/p>\n<p style=\"text-align: center;\"><i>Samples are indicated upon each band. C=control, 10=10\u00b5M, 100=100\u00b5M, 500=500\u00b5M of the beta-lactam antibiotic. After electrophoretic separation, the gel was directly placed on the UV-transilluminator (ESSENTIAL V6 System, UVITEC Ltd, UK) and activated for 60 seconds.<\/i><\/p>\n<p style=\"text-align: center;\"><i><img decoding=\"async\" src=\"https:\/\/biogroupvietnam.com\/assets\/uploads\/image\/picture4.png\" alt=\"\" width=\"300\" height=\"216\" \/><\/i><\/p>\n<p style=\"text-align: center;\"><i>Figure 2. Chemiluminescence Signal Of GLT-1 After ECL Reaction.<\/i><\/p>\n<p style=\"text-align: center;\"><i>Samples are indicated upon each band. C=control, 10=10\u00b5M, 100=100\u00b5M, 500=500\u00b5M of the beta-lactam antibiotic. The selected beta-lactam antibiotic increases the expression of GLT-1 in cultured astrocytes in a dose-dependent manner.<\/i><\/p>\n","protected":false},"excerpt":{"rendered":"<p>1. Introduction In Western blotting, normalization permits to mathematically correct the levels of a target protein to an internal control, which is used as indicator of sample protein loading. This procedure represents a critical step and it is required to correct for loading variation, thus confirming that the observed changes represent actual differences between samples.<\/p>\n<div class=\"klb-readmore post-buttons\"><a class=\"button rounded primary\" href=\"https:\/\/biogroupvietnam.com\/en\/new-products-technologies\/total-protein-normalization-stain-free-technology-and-uvitec-imagers\/\">Read More <i class=\"klbth-icon-right-arrow\"><\/i><\/a><\/div>\n","protected":false},"author":1,"featured_media":5631,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[17],"tags":[],"class_list":["post-4157","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-new-products-technologies"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.8 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Total protein normalization: Stain-Free technology and Uvitec imagers<\/title>\n<meta name=\"description\" content=\"1. Introduction In Western blotting, normalization permits to mathematically correct the levels of a target protein to an internal control, which is used as indicator of sample protein loading. 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