For the rapid, sensitive and accurate measurement of Citrate levels in various samples

Transcription

1 ab83396 Citrate Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Citrate levels in various samples This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis Troubleshooting 11 2

4 1. Overview Citric acid (HOOC-CH 2 -C(-OH)(-COOH)-CH 2 -COOH) is a key intermediate in the TCA cycle which occurs in mitochondria. It is formed by the addition of oxaloacetate to the acetyl group of acetyl- CoA derived from the glycolytic pathway. Citrate can be transported out of mitochondria and converted back to acetyl CoA for fatty acid synthesis. Citrate is an allosteric modulator of both fatty acid synthesis (acetyl-coa carboxylase) and glycolysis (phosphofructokinase). Citrate is widely used industrially in foods, beverages and pharmaceuticals. Citrate metabolism and disposition can vary widely due to sex, age and a variety of other factors. Abcam's Citrate Assay Kit provides a simple, sensitive and rapid means of quantifying citrate in a variety of samples. In the assay, citrate is converted to pyruvate via oxaloacetate. The pyruvate is quantified by converting a nearly colorless probe to an intensely colored (570 nm) and fluorescent (Ex/Em: 535/587 nm) product. The Citrate Assay Kit can detect 0.1 to 10 nmoles (~2 µm-10 mm) of citrate in a variety of samples. 3

5 2. Protocol Summary Sample Preparation Standard Curve Preparation Prepare and Add Reaction Mix Measure Optical Density or Fluorescence 4

6 3. Components and Storage A. Kit Components Item Quantity Citrate Assay Buffer 25 ml Citrate Probe 0.2 ml Citrate Enzyme Mix (Lyophilized) 1 vial Citrate Developer (Lyophilized) 1 vial Citrate Standard (10 µmol; Lyophilized) 1 vial Store kit at -20 C, protect from light. Warm Citrate Assay Buffer to room temperature before use. Briefly centrifuge all small vials prior to opening. Read the entire protocol before performing the assay. CITRATE STANDARD: Dissolve in 100 μl dh 2 O to generate 100 mm (100 nmol/μl) Citrate Standard solution. Keep on ice while in use. Store at -20 C. CITRATE PROBE: Ready to use as supplied. Warm to 37 C for 1 2 min to completely melt the DMSO solution before use. Protect from light. Store at -20 C. Use within 2 months. 5

7 CITRATE DEVELOPER, ENZYME MIX: Dissolve with 220 μl Assay Buffer separately. Pipette up and down to dissolve. Aliquot into portions, store at 20 C. Avoid repeated freeze/thaw cycles. Use within 2 months. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96-well plate Orbital shaker 6

8 4. Assay Protocol 1. Sample Preparation: Tissue (20 mg) or cells (2 x 10 6 ) should be rapidly homogenized with 100 μl of Citrate Assay Buffer. Centrifuge at 15,000 x g for 10 min to remove cell debris. Note: Enzymes in samples may interfere with the assay. We suggest deproteinizing samples using 10 kda molecular weight cut off spin columns (ab93349) or using a perchloric acid/koh protocol as follows: a) Tissue samples ( mg) should be frozen rapidly (liquid N 2 or methanol/dry ice), weighed and pulverized. b) Add 2 µl 1N perchloric acid/mg per sample. KEEP COLD! c) Homogenize or sonicate thoroughly. Spin homogenate at 10,000 x g for 5-10 minutes. d) Neutralize supernatant with 3M KHCO 3, adding repeated 1 µl aliquots/10 µl supernate while vortexing. Add until bubble evolution ceases (2-5 aliquots). Put on ice for 5 minutes e) Check ph (using 1 µl) is ~6-8. Spin 2 minutes at 10,000 x g to pellet KClO 4. Add 1-50 μl samples into duplicate wells of a 96-well plate and bring volume to 50 μl with Assay Buffer. 7

9 We suggest testing several doses of your samples to ensure readings are within the standard curve range. 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute the Citrate Standard to 1 nmol/μl by adding 10 μl of the Standard to 990 μl of dh 2 O, mix well. Add 0, 2, 4, 6, 8, 10 μl into a series of standards wells on a 96 well plate. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the Standard. b. For the fluorometric assay: Dilute the Citrate standard to 0.1 nmol/μl by adding 10 μl of the standard to 990 μl of dh 2 O, mix well, and then further dilute by adding 10 μl to 90 μl of dh 2 O. Add 0, 2, 4, 6, 8, 10 μl into a series of standards well on a 96-well plate. Adjust the volume to 50 μl/well to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well. 8

10 3. Citrate Reaction Mix: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 μl Reaction Mix containing: Colorimetric Assay Fluorometric Assay Sample Bkgd Control* Sample Bkgd Control* Assay Buffer 44 μl 46 μl 44 μl 46 μl Enzyme Mix 2 μl μl --- Developer 2 μl 2 μl 2 μl 2 μl Citrate Probe** 2 μl 2 μl 2 μl 2 μl * Note: Samples may contain oxaloacetate or pyruvate which can generate a background and need to be subtracted from the sample background signal. ** Note: For the fluorometric assay, dilute 10X with DMSO to reduce fluorescent background. 4. Add 50 μl of the Sample Reaction Mix to each well containing the Citrate Standard and Test Samples. Add 50 μl of the Background Control mix to Background Control wells. 5. Incubate for 30 min at room temperature, protect from light. 6. Measure OD at 570 nm or fluorescence at Ex/Em 535/587nm. 9

11 5. Data Analysis Correct background by subtracting the value of the zero Citrate Standard from all readings. Next subtract the value of the Sample Background Control from the test sample. Plot the standard curve. Apply corrected sample readings to the standard curve to get Citrate amount in the sample wells. The Citrate concentrations in the test samples: Concentration = Ay / Sv (nmol/μl; or μmol/ml; or mm) Where: Ay is the amount of citrate (nmol) in your sample from the standard curve. Sv is the sample volume (μl) added to the sample well. Citric acid molecular weight: 191 g/mol Citrate standard curve generated using this kit protocol 10

12 6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 11

13 Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 12

14 Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by (technical@abcam.com) or phone (select contact us on for the phone number for your region). 13

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16 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. 15 All information / detail is correct at time of going to print.