PicoProbe D-Lactate Assay Kit (Fluorometric)

Transcription

1 ab PicoProbe D-Lactate Assay Kit (Fluorometric) Instructions for Use For the sensitive and accurate measurement of D-Lactate in various samples This product is for research use only and is not intended for diagnostic use. Version: 1 Last Updated: 05 August 2013

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3 Table of Contents Table of Contents 2 1. Overview 3 2. Protocol Summary 4 3. Kits Components 5 4. Storage and Stability 5 5. Materials Required, Not Supplied 6 6. Reagents Preparation 7 7. Assay Protocol 8 8. Data Analysis Troubleshooting 16 2

4 1. Overview D-Lactate is the result of anaerobic glycolysis by microorganisms in the gastrointestinal system, and the product of detoxification of methylglyoxal by the glyoxalase system. The presence of abnormal levels of D-Lactate has been linked to a series of pathological conditions, such as diabetes, and appendicitis. Abcam's PicoProbe D-Lactate Assay Kit (Fluorometric) (ab174096) offers simplicity, sensitivity and can be adapted to high-throughput research. The assay enzymatically oxidizes D-Lactate generating a fluorescent signal (Ex/Em = 535/587 nm). The signal is directly proportional to the amount of D-Lactate. The assay kit can detect D-Lactate as low as 0.1 µm in a variety of samples Figure 1: Assay Procedure. 3

5 2. Protocol Summary Reagent Preparation Standard Curve Preparation Control & Samples Preparation Reaction Measurement and Calculation 4

6 3. Kits Components Item Quantity D-Lactate Assay Buffer 25 ml PicoProbe (in DMSO) 0.4 ml D-Lactate Enzyme Mix (Lyophilized) 1 vial D-Lactate Substrate Mix (Lyophilized) 1 vial D-Lactate Standard (100 mm) 0.1 ml 4. Storage and Stability Upon arrival, store the kit at -20 C and protect from light. Please read the entire protocol before performing the assay. Avoid repeated freeze/thaw cycles. Briefly centrifuge all small vials prior to opening. 5

7 5. Materials Required, Not Supplied 96-well black plate with flat bottom (for fluorometric reading) Multiwell spectrophotometer (ELISA reader) Multi-channel pipette MilliQ or double distilled water 6

8 6. Reagents Preparation 1. Picoprobe (in DMSO): Ready to use as supplied. Warm to room temperature before use to ensure DMSO is liquid. Store at -20 C. 2. D-Lactate Assay Buffer: Ready to use as supplied. Warm to room temperature before use. Store at -20 C. 3. D-Lactate Enzyme Mix: Reconstitute with 220 µl D-Lactate Assay Buffer. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Avoid repeated freeze/thaw. Keep on ice while in use. Stable for 2 months at -20 C. 4. D-Lactate Substrate Mix: Reconstitute with 220 μl D-Lactate Assay Buffer. Pipette up and down to dissolve completely. Aliquot and store at -20 C. Avoid repeated freeze/thaw. Keep on ice while in use. Stable for 2 months at -20 C. 5. D-Lactate Standard (100mM): Ready to use as supplied. Warm to room temperature before use. Store at -20 C. 7

9 7. Assay Protocol 1. Sample Preparation a) Cells (starting material: 2 x 10 6 cells) Harvest cells and spin down briefly and discard supernatant. Resuspend the cell pellet in 100 µl D-Lactate Assay Buffer and put on ice. Homogenize with a Douncer homogenizer (10 15 passes) on ice, or by pipetting up and down using a smaller tip, until efficient lysis is confirmed by viewing the cells under the microscope. Centrifuge homogenate at rpm for 10 minutes at 4 C to remove cell debris and collect the supernatant. Use the supernatant for your subsequent assays. Test different dilutions of the sample to ensure the readings will fall within the linear range of the standard curve. Add 2 50 µl test sample to wells of a 96-well plate. If volume needed is <50 µl, bring it up to 50 µl with Assay Buffer. For high target concentrations, dilute the samples to a total amount of 50 µl. b) Tissue(starting material: 20 mg) Cut tissue in small pieces, add 100 µl D-Lactate Assay Buffer and put on ice. Homogenize using a Douncer homogenizer (10 15 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the samples and collect the supernatant. 8

10 Use the supernatant for your subsequent assays. Test different dilutions of the sample to ensure the readings will fall within the linear range of the standard curve. Add 2 50 µl test sample to wells of a 96-well plate. If volume needed is <50 µl, bring it up to 50 µl with Assay Buffer. For high target concentrations, dilute the samples to a total amount of 50 µl. c) Body fluids (plasma, serum, urine and other biological fluids): Fluids should be filtered using a 10 kda MW spin filter (ab93349). Dilute samples with pure water (appropriate dilution factor is 1/10 1/100). Diluted samples can be assayed directly. Test different dilutions of the sample to ensure the readings will fall within the linear range of the standard curve. Add 2 50 µl test sample to wells of a 96-well plate. If volume needed is <50 µl, bring it up to 50 µl with Assay Buffer. For high target concentrations, dilute the samples to a total amount of 50 µl. d) Food samples. Beer: Remove CO 2 by vacuum-filtering samples. Dilute samples with pure water (appropriate dilution factor is approx 1/10). Diluted samples can be assayed directly. Test different dilutions of the sample to ensure the readings will fall within the linear range of the standard curve. 9

11 Yogurt: vortex 1 gram of yogurt in 10 ml of water until homogeneous. Dilute with pure water (appropriate dilution factor ~ 1/100 to 1/500). Diluted samples can be assayed directly. Test different dilutions of the sample to ensure the readings will fall within the linear range of the standard curve. Add 2 50 µl test sample to wells of a 96-well plate. If volume needed is <50 µl, bring it up to 50 µl with Assay Buffer. For high target concentrations, dilute the samples to a total amount of 50 µl. NOTE: Endogenous enzyme activity may cause loss of D- Lactate. Samples containing enzyme activity (such as culture medium or tissue lysate) should be kept at -80 C or filtered through a 10 kda MW spin filter. e) BACKGROUND CONTROL: For samples having high NADH levels, prepare a parallel sample well as the background control to avoid interference from NADH in the sample. 2. Standard Curve Preparation: a) Prepare a 1mM (1nmol/µl) D-Lactate Standard by adding 10 µl of 100 mm D-Lactate Standard to 990 µl D-Lactate Assay Buffer and mixing well. b) Prepare a 0.02 mm (20 pmol/µl) D-Lactate Standard by adding 20 µl of 1 mm Standard to 980 µl of D-Lactate Assay Buffer. 10

12 c) Using the 0.02 mm D-Lactate standard, prepare standard curve dilution as follows, in a microplate or microfuge centrifuge tubes: D-Lactate 0.02 mm D-Lactate Assay buffer Amount in well End concentration D-Lactate in well amount (µl) (µl) µl 0 pmol/well µl 40 pmol/well µl 80 pmol/well µl 120 pmol/well µl 160 pmol/well µl 200 pmol/well Add 50 µl of each standard dilution into a well in a 96-well plate to set up standard. Each dilution has enough amount of standard to set up 2 duplicates x 50 µl/well. 3. Reaction Mix: Prepare Reaction Mix for each reaction: Reaction Mix Background Control Mix D-Lactate Assay Buffer 45 µl 47 µl PicoProbe 1 µl 1 µl D-Lactate Enzyme Mix 2 µl ---- D-Lactate Substrate Mix 2 µl 2 µl 11

13 Mix enough reagents for the number of assays (samples and standards) to be performed. Prepare a Master Mix of the Reaction Mix to ensure consistency. We recommend the following calculation: Reaction Mix D-Lactate Assay Buffer 45 µl x (Nb of samples + Standards +1) PicoProbe 1 µl x (Nb of samples + Standards +1) D-Lactate Enzyme Mix 2 µl x (Nb of samples + Standards +1) D-Lactate Substrate Mix 2 µl x (Nb of samples + Standards +1) We also recommend preparing enough Background Control Mix to set up duplicate readings for the NADH background control using the same calculation. 4. Plate set up and Detection: a) Add 50 µl of standard and sample to wells. b) Add 50 µl of the Reaction Mix to each well containing standards and test samples. Mix well. c) Add 50 µl of Background Control Mix to 50 µl of background control wells(s). Mix well. d) Incubate the reaction for 30 minutes at 37 C, protected from light. e) Measure fluorescence (Ex/ Em = 535/587 nm) in a microplate reader with kinetics mode. 12

14 8. Data Analysis Calculations: a) Correct background by subtracting the value derived from the zero standard from all sample readings. b) Plot the D-Lactate Standard Curve. c) If Background Control reading is significantly high, subtract the Background Control reading from sample reading. Apply corrected sample reading to the D-Lactate Standard Curve to get B pmol of D-Lactate in the sample wells. Sample D-Lactate concentration = B/V x Dilution Factor = pmol/µl= nmol/ml = µm Where: B = the amount of D-Lactate in the sample well (pmol). V = volume of sample used in the reaction well (µl). D-Lactate in samples can also be expressed in nmol/mg of sample. D-Lactate molecular weight: g/mol. 13

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16 Figure 2. (a) D-Lactate Standard curve. The assay specifically detects D-Lactate, not L-Lactate (b) Measurement of D-Lactate in urine: Diluted samples (1/10, non-diabetic; 1/100 diabetic) were spiked with known amounts of D-Lactate (0-200 pmol) and assayed as specified. 15

17 9. 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 16

18 Problem Reason Solution 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) 17

19 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). 18

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