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Ab253372 Β-Glucuronidase (GUS) Reporter Gene Activity Detection Kit

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Ab253372 Β-Glucuronidase (GUS) Reporter Gene Activity Detection Kit Version 1 Last updated 14 June 2020 ab253372 β-Glucuronidase (GUS) Reporter Gene Activity Detection Kit View β-Glucuronidase (GUS) Reporter Gene Activity Detection Kit datasheet: www.abcam.com/ab253372 (use www.abcam.cn/ab253372 for China, or www.abcam.co.jp/ab253372 for Japan) For the measurement of β-Glucuronidase (GUS) activity in plant tissue. This product is for research use only and is not intended for diagnostic use. Copyright © 2020 Abcam. All rights reserved Table of Contents 1. Overview 1 2. Materials Supplied and Storage 2 3. Materials Required, Not Supplied 3 4. General guidelines, precautions, and troubleshooting 3 5. Reagent Preparation 4 6. Assay Procedure 5 7. Typical Data 7 8. Notes 9 Technical Support 10 Copyright © 2020 Abcam. All rights reserved 1. Overview Reporter genes are widely used as “markers” for analysis in gene regulation and localization, as well as for analysis of mutation altered genes. Expression of reporter genes can be measured by immunological assay, biochemical activity assay or by histochemical staining of cells or tissues. The β-glucuronidase (GUS) enzyme from E. coli (EC 3.2.1.31) has been well documented to provide desirable characteristics as a marker gene in transformed plants. The GUS reporter gene system has many advantages including stable expression of E. coli GUS enzyme, no interference with normal plant metabolism, and low intrinsic GUS activity in higher plants. The enzyme is also capable of tolerating amino-terminal additions, making it useful for study of plant organelle transport. Plants or other cell types are extracted with GUS extraction buffer. The extracted β-glucuronidase hydrolyzes the 4-MUG to the fluorescent compound 4-MU (pKa 8.2) and glucuronic acid. The reaction is stopped with sodium carbonate buffer because 4-MU exhibits maximal fluorescence at pH values above its pKa. 4-MU can be excited at 365nm and its emission maximum is 455nm. The GUS Reporter Gene Activity Detection Kit provides reagents, buffers, substrate, and protocols for sensitive and quantitative activity assays. Copyright © Abcam. All rights reserved 2. Materials Supplied and Storage Store kit immediately on receipt. Kit can be stored for 6 months from receipt, if components have not been diluted. High background fluorescence readings for blank samples will indicate decomposition. Aliquot components in working volumes before storing at the recommended temperature. Avoid repeated freeze-thaws of reagents. Storage Item Quantity temperature GUS Extraction Buffer 25 mL 4°C Carbonate Stop Buffer 25 mL 4°C GUS Assay Buffer 5 mL 4°C 4-MU calibration stock solution 5 mL 4°C Copyright © Abcam. All rights reserved 3. Materials Required, Not Supplied These materials are not included in the kit, but will be required to successfully perform this assay: UV-VIS Spectrometer or Plate reader. 96-well microplate Bradford Assay (to measure protein concentration) 4. General guidelines, precautions, and troubleshooting Please observe safe laboratory practice and consult the safety datasheet. For general guidelines, precautions, limitations on the use of our assay kits and general assay troubleshooting tips, particularly for first time users, please consult our guide: www.abcam.com/assaykitguidelines For typical data produced using the assay, please see the assay kit datasheet on our website. Copyright © Abcam. All rights reserved 5. Reagent Preparation - Equilibrate all reagents to room temperature prior to use. Adjust volumes as needed for your experiment. - Prepare only as much reagent as is needed on the day of the experiment. 5.1. 1X Carbonate Stop Buffer. Dilute provided Carbonate Stop Butter 1:5 in water to 0.2 M working concentration. 5.2 1X GUS Assay Buffer. Dilute 500 µL into 9.5 mL GUS Extraction Buffer to yield a 0.1mM 4-MUG solution for use in the assay. Specific conditions may require higher or lower concentrations, so dilute as necessary. 5.3 1µM MU Stock. Dilute 10µL of the 4-MU calibration stock solution into 10mL deionized water to make a 1µM 4-MU stock. Store at 4°C protected from light. Dilute this in 1X Carbonate Stop Buffer as needed to make 4-MU standard dilutions for use in extract activity calculations. Copyright © Abcam. All rights reserved 6. Assay Procedure Plant/Cell Extraction: This procedure may be modified or replaced with a different procedure as needed. 6.1 Weigh 5-50 mg plant tissue, add 200-500 µL of cold GUS Extraction Buffer, and grind with mortar and pestle until homogenized. 6.2 Place sample in microcentrifuge tubes and centrifuge for 8 minutes at 8000 rcf. Use supernatant to measure protein concentration using appropriate assay such as a Bradford assay. 6.3 Use the plant extract immediately or store frozen at -80°C(freezer or liquid nitrogen). Do not store the extract at -20°C; enzyme activity is lost at -20°C. NOTE: For other cell types (mammalian, bacterial, yeast, etc.), cell lysis conditions may vary. Consult with literature references for information about specific lysis conditions. Performing the assay: 6.4 To a 96-well microtiter plate, add the 1X GUS Assay Buffer, GUS Extraction Buffer, and plant extract, cell lysate or blank solution (extraction buffer) as described in the table below. 6.5 Allow at least 4 wells for each concentration of 4-MUG (two with plant extract or cell lysate and two with extraction buffer to serve as blanks and correct for any nonenzymatic hydrolysis of 4-MUG). Modify dilutions of 4-MUG and/or plant extract/cell lysate and conditions if necessary. For example, if a higher 4-MUG concentration is needed, use the 1X GUS Assay Buffer undiluted to give final [4-MUG] of 1.6mM, 1.2mM, 0.8mM, 0.4mM, and 0.2mM, respectively. 6.6 Incubate plate at 37°C for 10 minutes 6.7 Remove from heat and let sit at room temperature for 2-3 hours. 6.8 Add 200µL 0.2M Carbonate Stop Buffer to each well. 6.9 Measure fluorescence with emission and excitation filters set at 465nm and 360nm, respectively. Average the values and subtract the blank. To calculate extract activity it is necessary to perform the above assay using only one chosen concentration of 4-MUG, for example 1.6mM, where stop buffer is added to each of four wells (two with extract and two serving as blanks) at 20 minute intervals and fluorescence of Copyright © Abcam. All rights reserved those wells with stop buffer is measured and recorded. For the zero minute test points add stop buffer to wells immediately after addition of plant extract sample and record fluorescence. Average the values for each time interval and subtract the blank. Prepare 4-MU standard dilutions in 1X Carbonate Stop Buffer – concentrations of 10nM to 100nM are suitable. Include a minimum of 5 dilutions in order to plot a standard curve (10nm equals 20pmol in a 2mL cuvet). 6.10 Aliquot 200-300 µL of each dilution in duplicate into a microtiter plate and measure fluorescence. [4-MUG] GUS Assay GUS Enzyme or (Final) Buffer (0.1 mM Extraction Blank 4-MUG) Buffer Solution 0.08 mM 80 µL 10 µL 10 µL 0.06 mM 60 µL 30 µL 10 µL 0.04 mM 40 µL 50 µL 10 µL 0.02 mM 20 µL 70 µL 10 µL 0.01 mM 10 µL 80 µL 10 µL Table1. Sample Assay Conditions Copyright © Abcam. All rights reserved 7. Typical Data Data provided for demonstration purposes only. Figure 1. Assay performed with plant extract from flowers of Arabidopsis thaliana. Fluorescence was measured using a Perkin-Elmer HTS 7000 Plus UV/FL/LUM Microtiterplate reader. Copyright © Abcam. All rights reserved Calculations: Plot a calibration curve of 4-MU standards fluorescence intensity (FI) vs. pmol 4-MU. Plot a curve of sample FI vs. time. Calculate FI per pmol 4MU and FI per minute of extract sample. Calculate β-glucuronidase activity of extract in pmol 4-MU per minute per µg protein according to equation below. Sample calculation: 퐴푐푡푖푐푖푡푦 표푓 푒푥푡푟푎푐푡 = 퐹푙/푚푖푛 푟푒푎푐푡푖표푛 푣표푙 (µ퐿) 1 1 퐹푙/푝푚표푙 푀푈 × 푠푎푚푝푙푒 푣표푙 (µ퐿) × 푣표푙. 푝푒푟 푡푒푠푡 (µ퐿) × 푒푥푡푟푎푐푡 푐표푛푐 (µ푔 푝푟표푡푒푖푛/µ퐿) Figure 2. Comparison of GUS activity in leaf and flower extracts from Arabidopsis thaliana. Copyright © Abcam. All rights reserved 8. Notes Copyright © 2020 Abcam. All rights reserved Technical Support Copyright © 2020 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. For all technical or commercial enquiries please go to: www.abcam.com/contactus www.abcam.cn/contactus (China) www.abcam.co.jp/contactus (Japan) Copyright © 2020 Abcam. All rights reserved.
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