UC Davis - Uninary Albumin Excretion (UAE) Protocol Peter Havel1 May 15, 2019 1University of California, Davis 1 dx.doi.org/10.17504/protocols.io.yw5fxg6 Mouse Metabolic Phenotyping Centers Tech. support email: inf o@ mmpc. or g Lili Liang Summary: Albumin blue dye is a stain for the specific and sensitive spectrofluorometric determination of albumin in natural matrices. AB 580 binds to the albumin present in urine samples and the fluorescence can be quantified using a fluorimeter. DOI dx.doi.org/10.17504/protocols.io.yw5fxg6 https://mmpc.org/shared/document.aspx?id=98&docType=Protocol Peter Havel 2019. UC Davis - Uninary Albumin Excretion (UAE) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yw5fxg6 Albumin blue dye, Uninary Albumin Excretion (UAE) protocol , Mar 07, 2019 May 15, 2019 21181 1 Citation: Peter Havel UC Davis - Uninary Albumin Excretion (UAE) Protocol https://dx.doi.org/10.17504/protocols.io.yw5fxg6 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited MATERIALS Albumin Sigma Aldrich Catalog # A6414 Calibrator Diluent Sigma Aldrich Catalog # 09761 Albumin Blue 580 Potassium Salt Solution Sigma Aldrich Catalog # 05497 Buffer Sigma Aldrich Catalog # 79438 Microplate (for fluorescence) Contributed by users Fluorimeter Contributed by users Reagent Preparation: Standards – Dilute 10 mg of albumin with 5 ml of Calibrator Diluent to make a 2000 mg/ml stock. Then dilute the stock 1:9 by adding 20 μl of stock to 180 μl of Calibrator Diluent to make a 200 mg/ml standard. Dilute the 200 mg/ml standard 1:1 with Calibrator Diluent to make 100,50,25,12.5,6.25 standards. Calibrator Diluent – ready to use Working reagent – Mix 2 ml of Albumin Blue 580 Potassium Salt Solution with 100 ml of Buffer. Note: Sigma-Aldrich RRID:SCR_008988 1 Prepare working reagent. 2 Prepare standards by serially diluting 200 mg/l standard 1:1 to make 100, 50, 25, 12.5, 6.25 standards. 3 Add 25 μl of standard and sample to each well. 4 Add 125 μl of working reagent. Read in fluorimeter using 590 nm excitation and 616 nm emission. IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader. 2 Citation: Peter Havel UC Davis - Uninary Albumin Excretion (UAE) Protocol https://dx.doi.org/10.17504/protocols.io.yw5fxg6 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 5 Use a polynomial 2nd order curve fit to construct a standard curve. Interpolate the values of the unknowns using the standard curve. 3 Citation: Peter Havel UC Davis - Uninary Albumin Excretion (UAE) Protocol https://dx.doi.org/10.17504/protocols.io.yw5fxg6 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited