ab136957
Hexokinase Colorimetric
Assay Kit
Instructions for Use
For the sensitive and accurate measurement of
Hexokinase in serum, animal tissue and cell
culture samples (adherent and suspension)
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
10
6.
Troubleshooting
12
2
1. Overview
Hexokinases (HK) are found in many organisms including bacteria,
plants and mammals and play an important role in glucose
metabolism. Hexokinases phosphorylate glucose and generate
glucose-6-phosphate for glycolysis. Hexokinases have four isoforms
(HKI, II, III and IV). HK-I, HK-II and HK-III have low Km, while HK-IV
has 100 fold high Km. Hexokinase deficiency leads to severe human
diseases such as X-linked muscular dystrophy and a rare autosomal
recessive hemolytic anemia.
On the other hand, increased hexokinase activity is detected in
various human tumors and is associated with metastasis. Early
detection of abnormal hexokinase activity is crucial for diagnosis,
prediction and treatment of the disease.
In Abcam’s Hexokinase Assay kit, glucose is converted to glucose-6phosphate by hexokinase; the glucose-6-phosphate is oxidized by
glucose-6-phosphate dehydrogenase to form NADH, which reduces
a colorless probe to a colored product with strong absorbance at 450
nm. The assay is simple, sensitive and rapid and can detect
hexokinase activity even less than 0.1 mU/well.
3
2. Protocol Summary
Reagent Preparation
Standard Curve Preparation
Sample Preparation
Positive Control
Reaction Mix
Measurement and Calculation
4
3. Components and Storage
A. Kit Components
Item
Hexokinase Assay Buffer
Quantity
25 mL
Glucose
1 mL
ATP (Lyophilized)
1 vial
Enzyme Mix (Lyophilized)
1 vial
Developer (Lyophilized)
1 vial
NADH Standard (Lyophilized)
1 vial
Positive Control (Lyophilized)
1 vial
* Store the kit at-20°C and protect from light. Please read the entire
protocol before performing the assay. Avoid repeated freeze/thaw
cycles.
Warm all Buffers to room temperature before use. Briefly centrifuge
all small vials prior to opening.
5
B. Additional Materials Required

96-well clear plate with flat bottoms

Multi-well spectrophotometer (ELISA reader)
6
4. Assay Protocol
A. Reagent Preparation
1. ATP:
Reconstitute with 220 μl Assay Buffer to generate 0.2 M solution.
Store at -20°C. Use within two months. Keep on ice while in
use.
2. Enzyme Mix:
Reconstitute with 220 μl Assay Buffer. Pipette up and down to
dissolve completely. Aliquot and store at -20°C. Avoid
repeated freeze/thaw cycles. Use within two months. Keep on
ice while in use.
3. Developer:
Reconstitute with 220 μl dH2O. Pipette up and down to dissolve
completely. Store at -20°C. Use within two months.
4. NADH Standard:
Reconstitute
with
400
μl
dH2O
to
generate
1.25
mM
(1.25 nmol/μl) NADH Standard solution. Store at –20°C. Use
within two months. Keep on ice while in use.
5. Positive Control:
Reconstitute with 100 μl Assay Buffer and mix thoroughly.
Aliquot and store at –20°C.
7
B. Hexokinase Assay Protocol
1. NADH Standard Curve:
Add 0, 2, 4, 6, 8 and 10 μl of 1.25 mM NADH Standard into a
series of wells in duplicate in 96 well plate to generate 0, 2.5,
5.0, 7.5, 10 and 12.5 nmol/well of NADH Standard. Adjust
volume to 50 μl/well with Assay Buffer.
2. Sample Preparation:
Rapidly homogenize tissue (10 mg) or cells (1 x 106) with 200 μl
ice cold Assay Buffer for 10 minutes on ice. Centrifuge at 12000
rpm for 5 min. Collect the supernatant. Add 1-50 μl sample
(40 μg) per well, adjust final volume to 50 μl with Assay Buffer.
Prepare a parallel sample well as the background control to
avoid interference from the NADH in the sample.
Note: For unknown samples, we suggest testing several doses
to ensure the readings are within the standard curve range.
3. Positive Control:
Dilute Positive control solution 1:99 in Assay Buffer. Use 1-10 μl
of diluted Positive Control into the desired well(s) and adjust the
final volume to 50 μl with Assay Buffer.
8
4. Reaction Mix:
Mix enough reagents for the number of assays to be performed.
For each well, prepare 50 μl Mix containing:
Reaction Mix
Hexokinase Assay
Background
Control Mix
34 µl
44 µl
Enzyme Mix
2 µl
2 µl
Developer
2 µl
2 µl
ATP
2 µl
2 µl
Glucose
10 µl
---
Buffer
Add 50 μl of the Reaction Mix to each well containing the
Standard, Positive Control and test samples and 50 μl of
Background Control mix to each well containing the Background
Control sample. Mix well.
5. Measurement:
Incubate for 20-60 min at room temperature and measure
OD450nm.
Note: Incubation time depends on the Hexokinase activity in the
samples. We recommend measuring the OD in a kinetic mode,
and choose two time points (T1 & T2) in the linear range to
calculate the hexokinase activity of the samples. The NADH
standard curve can read in Endpoint mode (i.e., at the end of
incubation time).
9
5. Data Analysis
Calculation: Subtract the 0 standard reading from all standard
readings.
Plot
the
NADH
standard
curve.
Correct
sample
background by subtracting the value derived from the background
control from all sample readings. Calculate the hexokinase activity of
the test sample: ΔOD = A2 – A1. Apply the ΔOD to the NADH
standard curve to get B nmol of NADH generated by hexokinase
during the reaction time (ΔT = T2 - T1).
Sample Hexokinase
=
Activity
B
(ΔT x V)
x
Dilution
Factor
= nmol/min/ml/ = mU/ml
Where:
B is the NADH amount from standard curve (nmol).
ΔT is the reaction time (min).
V is the sample volume added into the reaction well (ml).
Unit Definition: One unit of hexokinase is the amount of enzyme that
will generate 1.0 μmol of NADH per min at pH 8 at room
temperature.
10
Figure 1: NADH standard curve (a). Hexokinase activity in Positive
Control and Jurkat cell lysate (40 μg) (b). Assays were performed
following kit protocol.
11
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
12
Problem
Reason
Solution
Samples
with
inconsistent
readings
Unsuitable sample
type
Refer to datasheet for details
about incompatible samples
Use the assay buffer provided
(or refer to datasheet for
instructions)
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
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
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 ([email protected]) or phone (select
“contact us” on www.abcam.com for the phone number for
your region).
14
UK, EU and ROW
Email: [email protected]
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: [email protected]
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: [email protected]
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: [email protected]
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
15
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.