ab102526 Lactate Dehydrogenase (LDH) Assay Kit (Colorimetric) Instructions for Use For the rapid, sensitive and accurate measurement of Lactate Dehydrogenase in various samples. This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 11 2
1. Overview Lactate dehydrogenase (LDH) is an oxidoreductase (EC 1.1.1.27) present in a wide variety of organisms. It catalyses the interconversion of pyruvate and lactate with concomitant inter-conversion of NADH and NAD +. When disease or injury or toxic material damages tissues, cells release LDH into the bloodstream. Since LDH is a fairly stable enzyme, LDH has been widely used to evaluate the presence of damage and toxicity of tissue and cells. Quantification of LDH has broad range of applications. In Abcam's Lactate Dehydrogenase (LDH) Assay Kit (Colorimetric), LDH reduces NAD to NADH, which then interacts with a specific probe to produce a color (λ max = 450 nm). The kit quantifies LDH activity in variety of biological samples such as in serum or plasma, cells, culture medium and fermentation, etc. The assay is quick, convenient, and sensitive. The kit can detect 1-100 mu/ml of LDH directly in samples. 3
2. Protocol Summary Sample Preparation Standard Curve Preparation Add Reaction Mix Measure Optical Density 4
3. Components and Storage A. Kit Components Item Quantity LDH Assay Buffer 50 ml LDH Substrate Mix (Lyophilized) 1 vial NADH Standard (0.5 μmol; Lyophilized) 1 vial LDH Positive Control 20 µl * Store kit at -20 C, protect from light. Warm the Assay Buffer to room temperature before use. Spin the vials prior to opening. Keep samples and LDH Positive Control on ice during the assay. All the solutions are stable for at least 1 week at 4 C and 1 month at -20 C. Read the entire protocol before the assay. SUBSTRATE MIX: Dissolve with 1 ml ddh 2 O for 10 min, sufficient for 500 reactions. 5
B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. Sample Preparation: a. Serum can be tested directly. b. Tissue cell or erythrocyte samples: Homogenize 0.1 g tissue, or 10 6 cells, or 0.2 ml Erythrocytes on ice in 0.5 ml cold Assay Buffer; Centrifuge at 10,000 x g for 15 min at 4 C; Collect the supernatant for assay and store on ice. Dilute LDH Positive Control 1:9 with Assay Buffer before use, and use 2-5 μl as Positive Control. Add 2-50 μl samples into a 96-well plate; bring the volume to 50 μl with Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. 2. NADH Standard Curve Preparation: Dissolve NADH Standard into 0.4 ml ddh 2 O to generate 1.25 mm NADH Standard Solution. Add 0, 2, 4, 6, 8, 10 μl of the 1.25 mm NADH Standard into 96-well plate in duplicate to generate 0, 2.5, 5.0, 7.5, 10.0, 12.5 nmol/well standard. Bring the final volume to 50 μl with Assay Buffer. 7
3. Reaction Mix: Mix enough reagents for the number of assays and standards to be performed. For each well, prepare a total 50 μl Reaction Mix: Assay Buffer 48 μl Substrate Mix Solution 2 μl Mix well. Add 50 μl of the Reaction Mix to each sample, Positive Control, and Standard, mix well. 4. Measure OD 450nm at T 1 to read A 1, measure OD 450nm again at T 2 after incubating the reaction at 37 C for 30 min (or longer if the LDH activity is low) to read A 2, protect from light. ΔA 450nm = A 2 A 1 Note: a) It is essential to read A 1 and A 2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A 1 and A 2 in the reaction linear range. b) For Standard Curve, use A 2 reading after 30 min incubation, do not subtract the A 1 reading. The Standard reading is stable for a few hours. 8
5. Data Analysis Subtract the zero nmol/well NADH background from all readings, plot NADH Standard Curve. Apply the sample ΔA 450nm to the NADH standard curve to get B nmol. Where: LDH Activity = B x Sample Dilution (T 2 T 1 ) x V = nmol/min/ml = mu/ml B is the NADH amount that was generated between T 1 and T 2 (in nmol). T 1 is the time of first reading (A 1 ) (in min). T 2 is the time of second reading (A 2 ) (in min). V is the pretreated sample volume added into the reaction well (in ml). NADH molecular weight: 763.0 g/mol. Unit definition: One unit LDH is the amount of enzyme that catalyzes the conversion of lactate to pyruvate to generate 1.0 μmol to NADH per minute at 37 C. 9
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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
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
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 email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 13
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