Research Areas > Mitochondrial Biogenesis > MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric)

MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric)

Catalog No. MS643

$425.00 - 2 x 96 tests


Description

For identifying inhibitors and activators of mitochondrial biogenesis in adherent cultured cells. Each kit contains sufficient reagents to analyze two 96-well plates of fixed human, rat, mouse, or bovine cells. This kit utilizes colorimetric detection for use with standard plate readers. An alternate IR version of this kit is available which utilizes LI-COR® near-infrared IRDyes® for detection - MitoBiogenesis™ In-Cell ELISA Kit (IR) (catalog # MS642).


Principle

In-Cell ELISA Kits use quantitative immunocytochemistry to measure protein levels or post-translational modifications in cultured cells. Cells are fixed in a 96-well plate and targets of interest are detected with highly specific, well-characterized monoclonal antibodies and levels are quantified with enzyme-labeled secondary antibodies.


Background

The MitoBiogenesis™ In-Cell ELISA Kit (catalog # MS643) is designed to measure drug-induced effects on mitochondrial biogenesis early in the safety screening process. The MitoBiogenesis™ In-Cell ELISA Kit is a true duplexing 96/384-well assay that ratios both an mtDNA- and an nDNA-encoded protein in cultured or primary cells, and which requires very little sample prep and few overall steps.

Cells (human, rat or mouse) are seeded in 96- or 384-well microplates, and after exposure to experimental compounds for several cell doublings, the levels of two mitochondrial proteins are measured simultaneously in each well. The two proteins are each subunits of a different oxidative phosphorylation enzyme complex, one protein being subunit I of Complex IV (COX-I), which is mtDNA-encoded, and the other being the 70kDa subunit of Complex II (SDH-A), which is nDNA-encoded. Complex IV includes several proteins which are encoded in the mitochondrion, while the proteins of Complex II are entirely encoded in the nucleus. Optionally, total protein levels can also be measured.



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Product Specifications
  
Applications: High-throughput analysis of mitochondrial biogenesis in adherent cells.
Species Reactivity: human, rat, mouse, bovine
Storage Conditions: Upon receipt spin down the contents of the IR-labeled secondary antibody tube. Store all components upright at 4°C. The kit is stable for at least 6 months.
Country of Origin: USA


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Specifications
 
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Figure 1


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Figure 1. Inhibition of mitochondrial biogenesis by chloramphenicol The IC50 of a drug's effect on mitochondrial protein translation can be determined quickly using the MitoBiogenesis™ In-Cell ELISA Kit. In this example, cells were seeded at 6000 cells/well, allowed to grow for 3 cell doublings in a drug dilution series and then the relative amounts of COX-I, and SDH-A were measured in each well. Chloramphenicol inhibits mtDNA-encoded COX-I protein synthesis relative to nuclear DNA-encoded SDH-A protein synthesis by 50% at 8.1 µM.
Figure 2a


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Figure 2a. Antibody specificity demonstrated by Western Blot. A Western blot of total cell protein (10 µg) from human or rat cultured cells was probed with the primary and secondary antibodies and scanned with a LI-COR® Odyssey® imager. The two mitochondrial proteins targeted by the two primary mAbs were labeled and visualized specifically despite the presence of thousands of other proteins. Furthermore, reduction of mtDNA levels in human Rho0 (mtDNA-depleted) cells, or inhibition of mitochondrial protein translation by chloramphenicol in rat cells both result in specific reduction of COX-I protein while nuclear DNA-encoded SDH-A is unaffected.

Figure 2b


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Figure 2b. Antibody specificity demonstrated by immunocytochemistry. Two-color immunocytochemical labeling of cultured cells with the two MS643 primary monoclonal antibodies specific for COX-I and SDH-A. The two antibodies exhibit striking and specific co-localization in the mitochondria, consistent with the known mitochondrial expression of both proteins.
Figure 3


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Figure 3. Quantitative measurement of the COX-I/SDH-A protein expression ratio. At all cell concentrations, a consistent ratio of mtDNA-encoded protein expression (COX-I) to nuclear DNA-encoded mitochondrial protein expression (SDH-A) is observed in untreated cells. Therefore, normalizing COX-I levels to SDH-A levels simplifies data analysis and eliminates the need to perform all tests at the same cell concentration.


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Specifications
 
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Downloadable Documents


   Technical Data Sheet

   Protocol

   Mitochondrial Biogenesis Playbook

   MSDS Sodium Azide

   MSDS Diethanolamine


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Specifications
 
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