ab110173 –
Frataxin Profiling ELISA
Kit
Instructions for Use
For the measurement of frataxin in Human samples of purified
mitochondria, cell and tissue extracts.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 19 December 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
4
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
5
5
5
6
6
7
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. SAMPLE PREPARATION
11. PLATE PREPARATION
8
9
11
ASSAY PROCEDURE
12.
ASSAY PROCEDURE
12
DATA ANALYSIS
13. TYPICAL DATA
14. SPECIES REACTIVITY
14
16
RESOURCES
15. FREQUENTLY ASKED QUESTIONS
16. NOTES
17
18
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1
INTRODUCTION
1. BACKGROUND
Abcam’s Frataxin in vitro Profiling ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed for the measurement of Frataxin
in Human samples of purified mitochondria, cell and tissue extracts.
Abcam’s Frataxin Profiling ELISA Kit is used to determine the amount
of Frataxin in a sample. Capture antibodies are pre-coated in the wells
of premium Nunc MaxiSorp™ modular microplates, which can be
broken into 8-well strips. Frataxin is immunocaptured within the wells.
The quantity of protein is measured by adding a second frataxin
specific antibody that is labeled with horseradish peroxidase. This
peroxidase changes the substrate from colorless to blue. The rate of
color development is proportional to the amount of protein captured in
the well and can be monitored at 600 nm. Alternatively the assay can
be terminated, at a user defined time, by the addition of 1N HCl (not
supplied) and the assay performed as an end point measurement at
450 nm.
Frataxin is encoded by the gene FXN on chromosome 9. Expression
has been shown in many tissues where the protein is imported into the
mitochondrial matrix and processed to a mature 17.3 kDa protein. The
role of Frataxin is not clear, however, it has been proposed to function
as an iron chaperone, an iron storage protein, as a repair enzyme of
damaged FeS clusters in aconitase, as a defense against oxidative
stress and activator of mitochondrial oxidative phosphorylation.
Partial frataxin deficiency in Humans results in Friedrich ataxia (FRDA)
an autosomal recessive disease characterized mainly by progressive
neurodegeneration in the spinal cord, and frequently by hypertrophic
cardiomyopathy and diabetes. FRDA is the most common form of
ataxia (1:30,000 - 1:50,000 in US and Europe). In greater than 95% of
cases the genetic basis of the deficiency is a triplet-nucleotide repeat
of GAA in intron 1 of both Frataxin alleles. Normal individuals carry
‘small number repeats’ (6-12) while less than 20% of normal
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2
INTRODUCTION
individuals contain ‘large normal repeats’ (14-34). The role of these
repeats, if any, is unknown. Individuals with large number repeats are
at increased risk of having offspring with expanded repeats (66-1500)
which result in disease. Expanded repeats interfere with transcription
of frataxin mRNA and result in a decrease in frataxin protein causing at
the cellular level impairment of energy metabolism, increased oxidative
stress, reduced heme biosynthesis, impaired iron metabolism and iron
accumulation in the heart and nervous system at the tissue level.
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3
INTRODUCTION
2. ASSAY SUMMARY
Remove appropriate number of
antibody coated well strips. Equilibrate
all reagents to room temperature.
Prepare all the reagents, samples, and
standards as instructed.
Add standard or sample to each well
used. Incubate at room temperature.
Aspirate and wash each well. Add
prepared Detector Antibody to each
well. Incubate at room temperature.
Aspirate and wash each well. Add
prepared HRP label. Incubate at room
temperature.
Aspirate and wash each well. Add
TMB Development Solution to each
well. Immediately begin recording the
color development. Alternatively add a
Stop solution at a user-defined time.
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GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at +2-8°C immediately upon receipt.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in the Reagent and Sample Preparation sections.
5. MATERIALS SUPPLIED
1 x 20 mL
Storage
Condition
(Before
Preparation)
+2-8°C
10X Blocking Buffer (Tube 2)
1 x 8 mL
+2-8°C
Development Solution (Tube 3)
1 x 20 mL
+2-8°C
Detergent
1 x 1 mL
+2-8°C
20X Detector Antibody (Tube A)
1 x 1 mL
+2-8°C
20X HRP Label (Tube B)
1 x 1 mL
+2-8°C
1 x 96 wells
+2-8°C
Item
20X Buffer (Tube 1)
96 – Well microplate (12 strips)
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Amount
5
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Microplate reader capable of measuring absorbance at 600 or
450 nm.

Method for determining protein concentration (BCA assay
recommended).

Deionized water.

Multi and single channel pipettes.

PBS (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM
KCl, pH 7.3).

Tubes for standard dilution.

Stop solution (optional) – 1N Hydrochloric acid (HCl).
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.
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6
GENERAL INFORMATION
8. TECHNICAL HINTS

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers.

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Complete removal of all solutions and buffers during wash steps is
necessary to minimize background.

As a guide, typical ranges of sample concentration for commonly
used sample types are shown below in Sample Preparation
(section 10).

All samples should be mixed thoroughly and gently.

Avoid multiply freeze/thaw of samples.

Incubate ELISA plates on a plate shaker during all incubation
steps (optional).

When generating positive control samples, it is advisable to
change pipette tips after each step.

This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that contain
sample, control or standard will vary by product. Review the
protocol completely to confirm this kit meets your
requirements. Please contact our Technical Support staff with
any questions.
or
bubbles
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when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents to room temperature (18-25°C) prior to use.
9.1
Solution 1
Prepare Solution 1 by adding 20 mL of 20X Buffer (Tube 1)
to 380 mL deionized H2O.
Store prepared Solution 1 at +2-8°C.
9.2
Incubation Solution
Prepare Incubation Solution by adding 8 mL of 10X Blocking
Buffer (Tube 2) to 72 mL of Solution 1 in a clean bottle or
tube.
Store prepared Incubation Solution at +2-8°C.
NOTE – When using fewer strips make proportionately less
(i.e. 670 μL Tube 2 + 6 mL Solution 1 for each strip used.
9.3
1X Detector Antibody (Solution A)
Prepare 1X Detector Antibody by diluting the Detector
Antibody 20-fold with Incubation Solution immediately prior
to use. When using all 12 strips add 1 mL of 20X Detector
Antibody (Tube A) to 20 mL of Incubation Solution. Mix
gently and thoroughly.
9.4
1X HRP Label (Solution B)
Prepare 1X HRP Label by diluting the 20X HRP Label 20fold with 1X Incubation Buffer immediately prior to use.
When using 12 strips, add 1 mL of 20X HRP Label (Tube B)
to 20 mL of Incubation Solution. Mix gently and thoroughly.
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ASSAY PREPARATION
10. SAMPLE PREPARATION
TYPICAL SAMPLE LINEAR RANGE Typical linear ranges
Sample Type
Human liver mitochondria
Whole cultured cell extract
Pure Frataxin (ab110353)
Range
0.3 – 10 μg/well
1 - 50μg/well
0.04 – 2.5 ng/well
1.5 – 50 μg/mL
5 – 250 μg/mL
0.2 – 12.5 ng/mL
Table 1. Typical linear ranges per well (200 μl) and per milliliter. The ranges
may be extended by using a non-linear fit of the data from a normal sample.
NOTE: Ranges for tissue extract may vary slightly. The lowest amount
indicated is the lowest amount tested (>4x background). For sample
loading use the recommended amount specified in this section.
Intra assay variation = 5.7% (n=96), inter assay variation = 12.5 %
(n=48)
This assay is designed for use with homogenates from cultured cells.
Isolated mitochondria and tissue lysates can also be but some sample
optimization may be necessary. As described below, homogenized
samples should be resuspended to 5.5 mg/mL protein. The proteins
are detergent extracted and loaded to within the linear range of the
assay (see below). A control or normal sample should always be
included in the assay as a reference. Also, include a null or buffer
control to act as a background reference measurement.
10.1 Determine the sample protein concentration by a standard
method and then adjust the protein concentration to
5.5 mg/mL in PBS.
NOTE – If the sample is less than 5.5 mg/mL, centrifuge to
pellet again and take up in a smaller volume to concentrate
the pellet and repeat protein concentration measurement.
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ASSAY PREPARATION
The optimal protein concentration for detergent extraction is
5.5 mg/mL.
10.2 Add 1/10 volume of Detergent to the sample (e.g. if the total
sample volume is 500 µL, add 50 µL of Detergent). The final
protein concentration is now 5 mg/mL.
10.3 Mix immediately and then incubate on ice for 30 minutes.
10.4 Spin in tabletop microfuge at maximum speed (~20,000 g,
~16,000 rpm) for 20 minutes. Carefully collect the
supernatant and save as sample. Discard the pellet.
10.5 The microplate wells are designed for 200 µL sample volume
per well, so dilute samples to the following recommended
concentrations by adding Incubation Solution (Note: Do not
use more than half the Incubation Solution prepared for
sample dilution):
Sample Type
Recommended amount
Whole cultured cell extract
10 μg/200 μL (50 μg/mL)
Pure Frataxin
1 μg/200 μL (2.5 ng/mL)
10.6 Keep diluted samples on ice until ready to proceed.
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ASSAY PREPARATION
11.PLATE PREPARATION

The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.

Unused well strips should be returned to the plate packet and
stored at 4°C.

For each assay performed, a minimum of 2 wells must be used as
the zero control.

For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).

Well effects have not been observed with this assay.
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ASSAY PROCEDURE
12. ASSAY PROCEDURE


Equilibrate all materials and prepared reagents to room
temperature prior to use.
It is recommended to assay all controls and samples in
duplicate.
12.1 Add 200 μL of each diluted sample into individual wells on
the plate. Include a normal sample as a positive control.
Include a buffer control (200 μL Incubation Solution) as a
null or background reference.
12.2
Incubate for 3 hours at room temperature.
12.3
The bound monoclonal antibody has immobilized the
protein in the wells. Empty the wells by turning the plate
over and shaking out any remaining liquid.
12.4
Once emptied, add 300 μL of Solution 1 to each well used.
12.5
Empty the wells again and add another 300 μL of Solution 1
to each well used.
12.6
Empty the wells again by turning the plate over and shaking
out the remaining liquid.
12.7
Add 200 μL of Solution A to each well used.
12.8
Incubate at room temperature for 1 hour.
12.9
Empty the wells again quickly by turning the plate over and
shaking out the remaining liquid.
12.10 Add 300 μL of Solution 1 to each well used. Empty the
wells again and add another 300 μL of Solution 1 to each
well used.
12.11 Empty the wells and add 200 μL of Solution B to each well
used.
12.12 Incubate at room temperature for 1 hour.
12.13 Empty the wells by turning the plate over and shaking out
any remaining liquid.
12.14 Once emptied, add 300 μL of Solution 1 to each well used.
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ASSAY PROCEDURE
12.15 Empty the wells again and add another 300 μL of Solution 1
to each well used. Repeat this step twice more for a total of
4 rinse steps.
12.16 Add 200 μL of Development Solution to each well used.
Any bubbles in the wells should be popped with a fine
needle as rapidly as possible.
12.17 Place the plate in the reader and record with the following
kinetic program (alternatively an endpoint measurement
can be made by stopping the reaction at a user defined
time by addition of 100 μL of 1N HCl per well).
Kinetic measurement
Endpoint Measurement
OD 600nm
Add 100µL 1M HCl/well
Time: 30 mins
Autoshake before readings
Interval: 20 sec – 1 min
Measure OD 450 nm
Autoshake between readings
12.18 Save data and analyze as described in the data analysis
section.
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13
DATA ANALYSIS
13. TYPICAL DATA

The quantity of frataxin captured in each well is proportional to the
amount of horseradish peroxidase activity within each well. The
quantity is the change in absorbance at 600 nm/minute/amount of
sample loaded into the well.

Examine the linear rate of increase in absorbance at 600 nm with
time. This is shown below where the rate/slope is calculated
between these time points. Most microplate software is capable of
performing this function. Repeat this for all samples.

For the control or normal sample, the rate versus amount loaded is
plotted as a straight line in the linear region of the assay as shown
below.

Compare the rates of the control (normal) sample and with the rate
of the null (background) and with your unknowns, experimental or
treated samples to get the relative amount of frataxin. When using
the pure recombinant frataxin (ab110353) compare the rates of
samples with the rates from diluted recombinant frataxin.
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DATA ANALYSIS
Figure 1. The change in absorbance is expressed as change in milliOD/min. In
the example above the change OD is divided by 1,000 and the time in seconds
is divided by 60 to obtain minutes.
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DATA ANALYSIS
14. SPECIES REACTIVITY
This kit detects Frataxin in Human samples only.
samples are not appropriate for use with this kit.
Mouse and rat
Other species have not been tested.
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RESOURCES
15. FREQUENTLY ASKED QUESTIONS
What is the minimum amount of cells or tissue needed to accurately
measure Frataxin quantity?
A signal of 4x over background was seen with 1 μg cell material per
well. This corresponds to approximately 10,000 cells per well.
Is it possible to speed up this assay?
Antigen-antibody reactions are dependent on many conditions such as
temperature and movement of molecules. The higher the temperature
and the faster the movement of molecules and the sooner the
saturation of binding sites occur. This assay can be performed in about
half the time if sample, detector and label incubations are carried out at
37°C on a rotating platform. However, it is crucial to be consistent with
all assays for cross-comparisons. Under these specified conditions,
samples can be incubated for 1.5 hours and detector and label
incubation times can be reduced to 35 minutes each.
Which immunogen was used to develop the antibodies used in this kit?
Recombinant Human Frataxin.
What evidence do you have that the captured protein is in fact pure
frataxin?
Immunoprecipitations using these antibodies were performed on a
large scale and analyzed by SDS-PAGE purity and Mass Spectrometry
for identity.
Which is the exact epitope that binds to the capture antibody (attached
to the plate) and the detector antibody provided with this kit?
The exact epitopes are unknown.
How do I analyze tissue samples?
Tissue samples must be homogenous before detergent extraction.
Homogenize tissue samples thoroughly by using a Dounce
homogenizer or microtissue grinder. Measure protein concentration by
BCA protein assay. Dilute to 5.5 mg/ml in PBS, then add 1/10 volume
of detergent for extraction and proceed from Sample Preparation step
10.3.
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RESOURCES
16. NOTES
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