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.

  Figure 2. Detailed overview of antibody interactions.


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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