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

Transcription

1 ab65330 L Lactate Assay Kit (Fluorometric) Instructions for Use For the rapid, sensitive and accurate measurement of Lactate levels in various samples This product is for research use only and is not intended for diagnostic use. Version: 5 Last Updated: 9 June 2014

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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 12 2

4 1. Overview Abnormally high concentrations of lactate have been related to disease states such as diabetes and lactate acidosis, etc. L(+)-Lactate is the major stereo-isomer of lactate formed in human intermediary metabolism and is present in blood. D(-)-Lactate is also present but only at about 1-5% of the concentration of L(+)-Lactate. In Abcam s L Lactate Assay Kit (Fluorometric), lactate specifically reacts with an enzyme mix to generate a product, which interacts with lactate probe to produce color (570 nm) and fluorescence (at Ex/Em = 535/587 nm). The kit provides a convenient means for detecting L(+)-Lactate in biological samples such as in blood circulation, in cells, in culture mediums, in fermentation mediums, etc. There is no need for pre-treatment or purification of samples. The kit can detect mm of various Lactate samples. 3

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

6 3. Components and Storage A. Kit Components Item Quantity Lactate Assay Buffer 25 ml Lactate Probe ml Lactate Enzyme Mix 1 vial 100 nmol/µl L(+)-Lactate Standard 100 µl * Store kit at -20 C. LACTATE ASSAY BUFFER: Ready to use as supplied. Allow to warm to room temperature before use. Store at -20 C. LACTATE PROBE: Ready to use as supplied. Allow to warm to room temperature to thaw the DMSO solution before use. Store at -20 C, protect from light. Use within two months. LACTATE ENZYME MIX: Dissolve in 220 μl Lactate Assay Buffer. Pipette up and down to completely dissolve. Store at -20 C. Use within two months. 5

7 LACTATE STANDARD: Ready to use as supplied. Allow to warm to room temperature before use. Store at -20 C. 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: Prepare test samples in 50 μl/well with Lactate Assay Buffer in a 96-well plate. If using serum sample, serum ( μl/assay, serum contains ~0.6 nmol/μl lactate) can be directly diluted in the Lactate Assay Buffer. Notes: a) Complete medium containing FBS should be deproteinized (see below) due to high LDH content. We suggest using several doses of your sample to ensure the readings are within standard curve range. For samples having background, prepare parallel well(s) containing same amount of sample as in the test well as backgroundcontrol. Endogenous compounds may interfere with the reaction. To ensure accurate determination of Lactate in the test samples, we recommend spiking samples with a known amount of Standard (4 nmol). b) Lactate Dehydrogenase (LDH) will degrade lactate. Therefore, samples containing LDH (such as culture medium or tissue lysate) should be kept -80 C for storage, or filter samples through 10 kda molecular weight spin filter (ab93349). 7

9 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute the Lactate Standard (MW 90.08) to 1 nmol/μl by adding 10 μl of the Lactate Standard to 990 μl of Lactate Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Lactate Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the L(+)- Lactate Standard. b. For the fluorometric assay: Dilute the Lactate Standard (MW 90.08) to 1 nmol/μl by adding 10 μl of the 100 nmol/μl Lactate Standard to 990 μl of Lactate Assay Buffer, mix well. Add 0, 2, 4, 6, 8 & 10 μl into a series of wells. Adjust the volume to 50 μl/well with Lactate Assay Buffer to generate 0, 2, 4, 6, 8 & 10 nmol/well of the L(+)-Lactate Standard. For fluorometric assay, dilute the Lactate Standard to 0.01 nmol/μl by adding 10 μl of the 1 nmol/μl Lactate Standard to 990 μl of Lactate Assay Buffer, mix well. Add 0, 2, 4, 6, 8 & 10 μl of 0.01 nmol/μl Lactate Standard into a series of wells. Adjust the volume to 50 μl/well with Lactate Assay Buffer to generate 0, 20, 40, 60, 80 & 100 pmol/well of the Lactate Standard. 8

10 3. Lactate Reaction Mix: Mix enough reagent for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix containing the following components: Lactate Assay Buffer 46 μl Probe 2 μl Enzyme Mix 2 μl Mix well before use. Add 50 μl of the Reaction Mix to each well containing the Lactate Standard or test samples, mix well. For samples having high background, add 50 μl of Background Control Mix (48 μl Assay Mix and 2 μl of Probe) to sample background control well(s). 4. Incubate the reaction for 30 minutes at room temperature, protect from light. 5. Measure OD at 570 nm for colorimetric assay or fluorescence at Ex/Em = 535/590 nm in a microplate reader. If the background is too high in the fluorometric assay, 1/10 volume of probe may be used, which will decrease the background significantly. 9

11 5. Data Analysis Correct background by subtracting the value derived from the zero lactate control from all sample readings. The background reading can be significant and must be subtracted from sample readings. Plot standard curve nmol/well ~OD 570nm readings. Apply the sample readings to the standard curve. Calculate the lactate concentrations of the test samples: Concentration = La / Sv (nmol/μl or mm) Where: La is the lactate acid amount (nmol) of your sample from standard curve. Sv is the sample volume (μl) added into the well. Lactate acid molecular weight: Lactate acid concentration in your sample can be expressed by the way of your choice. 10

12 Lactate Standard Curve performed according to assay protocol. Lactate Standard Curve performed according to assay 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 (technical@abcam.com) 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 technical@abcam.co.jp Tel: +81-(0) Copyright 2014 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.