MitoSciences Dipstick Assay Technology
MitoSciences MitoProfile® Dipstick Assay
technology is a fast, reproducible, quantitative method for measuring
protein quantity or enzyme activity from a wide range of sample
sources. Measurements can be obtained from cell culture samples such as
fibroblasts, PBMCs, lymphoblastoids, and various tissue types.
Products Highlights:
• SPEED: Time required to perform a dipstick assay is between 30 and
120 minutes.
• SIMPLICITY: Assay is easy to perform and sample preparation is
minimal as isolation of mitochondria is not necessary.
• SENSITIVITY: Sample requirement is as little as a few micrograms of
whole cell or tissue extract.
• VERSATILITY: The dipstick assays can be used for analysis of multiple
types of sample material.
I. Dipstick Assay for Protein Quantity
The dipstick assays for protein quantity utilize a binding scheme
common to protein sandwich assays using two monoclonal antibodies that
recognize different epitopes on a target protein or enzyme complex. One
antibody is embedded on the nitrocellulose membrane of the dipstick
while the other is conjugated to gold particles (Au). The signal
intensity correlates to the enzyme quantity and is measured by a
dipstick reader (MitoSciences'
Hamamatsu ICA-1000) or by any other imaging system
such as a flat-bed scanner.
Figure 1 is an illustration of a dipstick after signal development. The
upper band is the positive control signal and the lower band is the
target protein signal. The intensity of the signal correlates directly
with the protein levels present in the sample. Figure 2 is a schematic
of the gold-conjugated antibody binding to the captured target protein.
Figure
1. Schematic
representation of a dipstick.
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Figure 2. Detailed overview of
antibody interactions.
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II. Dipstick Assay for Enzyme Activity
The dipstick assays for enzyme activity are based on measuring the
activity of an immunocaptured enzyme directly on the dipstick. An
immobilized capture antibody on the dipstick binds the enzyme and
activity is measured by providing enzyme substrates and a precipitating
reporter dye for signal visualization. The signal intensity correlates
to the enzyme activity level and is measured by a dipstick reader (MitoSciences' Hamamatsu ICA-1000)
or by any other imaging system such as a flat-bed scanner.
A. Measuring Complex I activity
Complex I is immunocaptured onto the dipstick. The dipstick is immersed
in Complex I activity buffer containing reduced NADH as a substrate and
nitrotetrazolium (NBT) as the electron acceptor (reporter dye).
Immunocaptured Complex I oxidizes NADH; the resulting H+
reduces NBT to form a blue-purple precipitate on the dipstick.
B. Measuring Complex IV (COX) Activity
The COX enzyme complex is immunocaptured on the dipstick. The dipstick
is immersed in COX activity buffer containing reduced cytochrome c
and di-amino benzidinetetrachloride (DAB) as a reporter dye.
Immunocaptured COX oxidizes cytochrome c, which in
turn oxidizes DAB to form a reddish brown precipitate on the dipstick.
C. Measuring PDH Activity
The PDH complex is immunocaptured on the dipstick. The dipstick is
immersed in PDH activity buffer containing pyruvate TPP, Coenzyme A,
NAD+ and nitrotetrazolium (NBT). PDH reduces NAD+ to NADH, which is
coupled to the reduction of NBT to NBTH, forming a blue-purple
precipitate on the dipstick.
Dipstick Assay Kits
MS130
Dipstick Assay Kit for Complex I
Activity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of NADH ubiquinone oxidoreductase activity in
human, bovine, rat, and mouse samples.
MS131
Dipstick Assay Kit for Human
Complex I Quantity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of NADH ubiquinone oxidoreductase in human and
bovine samples.
MS133
Dipstick Assay Kit for Rodent
Complex I Quantity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of NADH ubiquinone oxidoreductase in mouse and
rat samples.
MS430
Dipstick Assay Kit for Human
Complex IV Activity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of cytochrome c oxidase activity in human and
bovine samples.
MS431
Dipstick Assay Kit for Human
Complex IV Quantity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of cytochrome c oxidase in human and bovine
samples.
MS432
Dipstick Assay Kit for Rodent
Complex IV Activity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of cytochrome c oxidase activity in mouse and rat
samples.
MS631
MitoProfile® Biogenesis Dipstick Assay Kit
MS631 is a rapid and simple test for determining whether mitochondrial
biogenesis has been up-regulated or down-regulated in human samples.
The assay works on very small amounts of sample material, including
cheek swabs or single drops of blood, and results can be generated in
under 30 minutes.
MS741
Dipstick Assay Kit for Human
Mitochondrial Trifunctional Protein (TFP)
Contains 30 or 60 dipsticks and necessary components to quantify the
amount of the Mitochondrial Trifunctional Protein (TFP) from human
sample material.
MSA31
Dipstick Assay Kit for
Apoptosis-Inducing Factor (AIF) Quantity
This kit contains 30 or 60 dipsticks and necessary components to
quantify the amount of Apoptosis-Inducing Factor (AIF) from human
sample material.
MSF31
Dipstick Assay Kit for Frataxin
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of human frataxin protein levels.
MSF42
Recombinant Human Frataxin
Frataxin is a nuclear-encoded mitochondrial protein. In humans the gene
is localized on chromosome 9 and is highly conserved during evolution.
The gene is expressed in every cell, although in varying levels in
different tissues and during development. Friedreich's Ataxia (FRDA) is
an inherited recessive neurodegenerative disorder caused by decreased
expression levels of frataxin. FRDA is the most common inherited ataxia
and it causes progressive damage to the nervous system, resulting in
symptoms ranging from muscle weakness and speech problems to heart
disease. About one in 50,000 people in the United States have
Friedreich's Ataxia.
MSP30
Dipstick Assay Kit for PDH Activity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of PDH activity in human, bovine, mouse, rat, and
pig samples.
MSP31
Dipstick Assay Kit for PDH Quantity
This kit contains 30 or 60 dipsticks and necessary components for
quantitative analysis of PDH in human, bovine, mouse, and rat samples.
Immunochromatographic Reader
While visual measurements and scanning on a flatbed scanner can both be
used to evaluate our dipsticks, the most effective quantitative results
are derived when using an immunochromatographic reader that has been
specifically designed to read conjugated gold lateral flow devices.
The reader that we recommend is the Hamamatsu
ICA-1000, which is a simple PC-based system for
measuring and analyzing our dipstick products. We have an agreement in
place with Hamamatsu that allows us to offer this reader directly to
our customers. To learn more about this reader, or to order one, please
click
here to visit the product page.
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