Abcam’s MitoBiogenesis™ Immmunocytochemistry kit is designed for to evaluate drug-induced effects on mitochondrial biogenesis early in the safety screening process. The assay allows for imaging and qualitative analysis between mtDNA encoded protein MTCO1 and nDNA encoded SDHA proteins in cells.
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 70 kDa 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.
The MitoBiogenesis™ Immunocytochemistry kit enables researchers to evaluate in-vivo levels of target proteins detected by highly specific, well-characterized monoclonal antibodies that are labeled with Alexa® fluorophores. Thus, minimizing potential changes during sample preparation and handling, such as preparation of protein extracts.
The Alexa Fluor® dye included in this product is provided under an intellectual property license from Life Technologies Corporation. As this product contains the Alexa Fluor® dye, the purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). As this product contains the Alexa Fluor® dye the sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are sold for use in research. For information on purchasing a license to use products containing Alexa Fluor® dyes for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or firstname.lastname@example.org
|100X MTCO1 AlexaFluor® 488 & SDHA AlexaFluor® 594 Cocktail||1 x 250µl|
|100X Triton X-100||1 x 500µl|
|10X Phosphate Buffered Saline||1 x 100ml|
|Antigen Retrieval Buffer||1 x 25ml|
|Blocking Solution||1 x 10ml|
Our Abpromise guarantee covers the use of ab170194 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration.|
Cells were treated with 10 µM chloramphenicol for a period of 6 days. Results of immunostaining of HeLa cells showing MTCO1 Alexa®488 antibody (ab154477, green) on untreated (A) and chloramphenicol treated (B). SDHA Alexa®594 antibody (red) on untreated (C) and chloramphenicol treated (D). DAPI staining of nucleus (blue) on untreated (E) and chloramphenicol treated (F). Merge of color channels for untreated (G) and chloramphenicol treated (H) cells.
Immunocytochemistry with HeLa cells (100x). A) HeLa stained with anti-SDHA Alexa® 594 antibody (1.0 µg/mL). B) HeLa stained with Anti-HSP60 (1/1000, ab46798), Secondary antibody used was goat anti-rabbit Alexa® 488 (1/1000, ab150077). C) DAPI as nuclear stain (1/10000). D) Merge of color channels to show specificity of signal to mitochondria.
A total of 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 antibodies 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 result in specific reduction of COX-I protein while nuclear DNA-encoded SDH-A is unaffected.
A) Staining of HeLa cells with Immunocytochemistry with HeLa cells (100x) were stained with anti-MTCO1 Alexa® 488 antibody (1.0 µg/mL, ab154477) in green and DAPI in blue, as a nuclear stain. B) Immunocytochemistry with HDFn (100x) cells were stained with Anti-MTOC1 Alexa® 488 antibody (1.0 µg/mL, ab154477) in green, Anti-HSP60 (1/1000, ab46798) as red, and DAPI in blue, as a nuclear stain. Secondary antibody used was goat anti-rabbit dyelight-594 (1/1000, ab96897).
ab170194 has not yet been referenced specifically in any publications.