PicoProbe Acetyl CoA Assay Kit

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1 ab87546 PicoProbe Acetyl CoA Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Acetyl CoA levels in various samples. This product is for research use only and is not intended for diagnostic use.

2 Table of Contents 1. Overview 2 2. Protocol Summary 3 3. Components and Storage 4 4. Assay Protocol 6 5. Data Analysis Troubleshooting 12 1

3 1. Overview Acetyl CoA is a central molecule of metabolism. It carries acetate, used in the build-up and breakdown of larger molecules. Acetyl CoA is key in synthetic pathways leading to sesquiterpenes, precursors to cholesterol and other sterols, flavenoids and other polyketides, polyenes and long-chain fatty acids. It is the source of the acetyl group used in histone acetylation. The acetyl group is also incorporated into a variety of other molecules such as acetylcholine, melatonin, heme and TCA cycle intermediates. Abcam s PicoProbe Acetyl CoA Assay Kit is a highly sensitive assay for determining Acetyl CoA level in a variety of biological samples. In the assay, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with PicoProbe to generate fluorescence (Ex=535/Em=587 nm). The assay can detect pmol of Acetyl CoA (with detection limit ~0.4 µm) in a variety of samples. 2

4 2. Protocol Summary Standard Curve Preparation Sample Preparation A CoA Conversion Add Reaction Mix Measure Fluorescence 3

5 3. Components and Storage A. Kit Components Item Quantity Acetyl CoA Assay Buffer 25 ml PicoProbe 0.2 ml Conversion Enzyme 0.1 ml Acetyl CoA Enzyme Mix 0.5 ml Acetyl CoA Substrate Mix (Lyophilized) 1 vial CoA Quencher 1 ml Quench Remover (Lyophilized) 1 vial Acetyl CoA Standard (10 µmol, Lyophilized) 1 vial * Store kit at -20 C, protect from light. Warm Acetyl CoA Assay Buffer to room temperature prior to using it. Briefly centrifuge all small vials prior to opening. Read the entire protocol before performing the assay. 4

6 PICOPROBE: Shipped in DMSO, ready to use as supplied. Thaw by warming to room temperature. Mix well, store at -20 C. ACETYL COA SUBSTRATE MIX: Dissolve Substrate Mix with 220 µl Assay Buffer. Pipette up and down to completely dissolve. Once reconstituted, store at -20 C. Use within two months. ACETYL COA STANDARD: Dissolve Acetyl CoA Standard in 100 µl dh 2 O to generate 10 mm (10 nmol/µl) Acetyl CoA Standard solution. Keep cold while in use. Once reconstituted, store at -20 C. QUENCH REMOVER: Dissolve Quench Remover in 220 µl dh 2 O. Keep on ice while in use, store at -20 C. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 5

7 4. Assay Protocol 1. Standard Curve Preparation: a. 0-1 nmol Range: Dilute the Acetyl CoA Standard 100X to 0.1 mm (100 pmol/µl) by taking 10 µl into 990 µl dh 2 O. Dilute a further 5X to 0.02 mm by adding 100 µl to 400 µl dh 2 O. Add 0, 10, 20, 30, 40, 50 µl into a series of wells in a 96-well plate. Adjust volume to 50 µl/well with dh 2 O to generate 0, 200, 400, 600, 800, 1000 pmol/well Acetyl CoA standard. b pmol Range: Dilute the Acetyl CoA Standard 100X to 0.1 mm (100 pmol/µl) by taking 10 µl into 990 µl dh 2 O. Dilute an additional 50X to 2 µm (2 pmol/µl) by taking 10 µl into 490 µl of dh 2 O. Mix well. Add 0, 10, 20, 30, 40, 50 µl into a series of standards wells on a 96-well plate. Adjust volume to 50 µl/well with dh 2 O to generate 0, 20, 40, 60, 80, 100 pmol/well Acetyl CoA standard. 6

8 2. Sample Preparation: Preparation of Cell Culture Samples: Cell Lysis Buffer (1X): 20 mm Tris (ph 7.5) 150 mm NaCl 1 mm EDTA 1 mm EGTA 1% Triton X mm sodium pyrophosphate 1 mm b-glycerolphosphate 1 mm Na3VO4 1 µg/ml Leupeptin Note: We recommend adding 1 mm PMSF before use. Preparing Cell Lysates a) Aspirate media. Treat cells by adding fresh media containing regulator for desired time. b) To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS. c) Remove PBS and add 0.5 ml 1X ice-cold Cell Lysis Buffer plus 1 mm PMSF to each plate (10 cm2) and incubate the plate on ice for 5 minutes. 7

9 d) Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice. e) Sonicate 4 times for 5 seconds each on ice. f) Microcentrifuge for 10 minutes at 4 C, and transfer the supernatant to a new tube. The supernatant is the cell lysate. g) If necessary, lysate can be stored at 80 C. h) Once the lysate is ready, follow the deproteinization protocol as mentioned below and then proceed with the whole protocol. Deproteinization protocol: Enzymes in samples interfere with the assay. You should deproteinize your sample 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 8

10 e) Check ph (using 1 µl) is ~6-8. Spin 2 minutes at 10,000 x g to pellet KClO 4. Add 10 µl samples into duplicate wells (Sample and Background) of a 96-well plate; bring volume to 50 µl with Assay Buffer. 3. Free CoASH and succ-coa in samples generate background. In order to correct for this background, add 10 µl of CoASH Quencher to each Standard, Sample and background sample to quench free CoA. Incubate for 5 min at room temp. Then add 2 µl of Quench Remover, mix and incubate 5 min. In addition, run background control for each sample to correct for succ-coa or some other forms by omitting the Conversion Enzyme. 4. CoA Conversion: Make up 50 µl of reaction mix for each well to be tested (Standard, Sample and Background): 0.1 nmol Bkgd pmol Bkgd Buffer 40 µl 41 µl 41.8 µl 42.8 µl Substrate Mix 2 µl 2 µl 2 µl 2 µl Conversion Enzyme 1 µl µl --- Enzyme Mix 5 µl 5 µl 5 µl 2 µl PicoProbe 2 µl 2 µl 0.2 µl 0.2 µl Mix well. Incubate at 37 C for 10 minutes 9

11 5. Measure fluorescence using Ex/Em = 535/589 nm with a plate reader. 5. Data Analysis Correct background by subtracting the value of the zero Acetyl CoA Standard from all readings. The background reading can be significant and must be subtracted from sample readings. Determine Background values for each sample tested and correct Acetyl CoA values for this background. Plot the Standard Curve. Apply the sample readings to the Standard Curve to get the Acetyl CoA amount in the sample wells. The Acetyl CoA concentrations in the test samples: Concentration = Ay / Sv (pmol/µl; or nmol/ml; or µm) Where: Ay is the amount of Acetyl CoA (pmol) in your sample from the Standard Curve. Sv is the sample volume (µl) added to the sample well. Acetyl CoA molecular weight:

12 Standard curves were generated following the kit protocol. 11

13 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 12

14 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) 13

15 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 or phone (select contact us on for the phone number for your region). 14

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 Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 15

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