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.
1
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 (λmax = 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
LDH Assay Buffer
Quantity
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 ddH2O 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 106 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 ddH2O 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
OD450nm at T1 to read A1, measure OD450nm again at T2 after
incubating the reaction at 37°C for 30 min (or longer if the LDH
activity is low) to read A2, protect from light.
ΔA450nm = A2 – A1
Note:
a) It is essential to read A1 and A2 in the reaction linear range. It will
be more accurate if you read the reaction kinetics. Then choose
A1 and A2 in the reaction linear range.
b) For Standard Curve, use A2 reading after 30 min incubation, do
not subtract the A1 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 ΔA450nm to the NADH standard curve to get B nmol.
LDH Activity =
B x Sample Dilution
= nmol/min/ml = mU/ml
(T2 – T1) x V
Where:
B is the NADH amount that was generated between T1 and T2
(in nmol).
T1 is the time of first reading (A1) (in min).
T2 is the time of second reading (A2) (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
10
6. Troubleshooting
Problem
Reason
Solution
Assay not
working
Assay buffer at
wrong temperature
Assay buffer must not be chilled
- needs to be at RT
Protocol step missed
Plate read at
incorrect wavelength
Unsuitable microtiter
plate for assay
Unexpected
results
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
Measured at wrong
wavelength
Use appropriate reader and filter
settings described in datasheet
Samples contain
impeding substances
Unsuitable sample
type
Sample readings are
outside linear range
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
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
Lower/
Higher
readings in
samples
and
standards
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
Wait for components to thaw
completely and gently mix prior
use
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
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
14
UK, EU and ROW
Email: technical@abcam.com
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: us.technical@abcam.com
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: hk.technical@abcam.com
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: technical@abcam.co.jp
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.