MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric) (ab110217)

製品の概要

  • 製品名
    MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric)
    MitoBiogenesis キット 製品一覧
  • 検出方法
    Colorimetric
  • サンプルの種類
    Adherent cells
  • アッセイタイプ
    Cell-based (quantitative)
  • ステップ
    Multiple steps standard assay
  • 種交差性
    交差種: Mouse, Rat, Cow, Human
  • 製品の概要

    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) (ab110216/MS642).

     

    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.

     

    ab110216 MitoBiogenesis™ In-Cell ELISA Kit (ab110216/MS643) is designed to measure drug-induced effects on mitochondrial biogenesis early in the safety screening process. ab110216 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.

     

    LI-COR®, Odyssey®, Aerius® and IRDye® are registered trademarks of LI-COR Biosciences Inc

     

    Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.

  • アプリケーション
    適用あり: In-Cell ELISAmore details
  • 試験プラットフォーム
    Microplate

製品の特性

  • 保存方法
    Store at +4°C. Please refer to protocols.
  • 内容 2 x 96 tests
    100X Triton X-100 1 x 1.5ml
    10X Blocking Buffer 1 x 15ml
    10X Phosphate Buffered Saline 1 x 100ml
    1X AP Development Solution 1 x 24ml
    1X HRP Development Solution 1 x 24ml
    200X Primary Antibodies 1 x 0.1ml
    2500X AP-labeled Secondary Antibody 1 x 12µl
    2500X HRP-labeled Secondary Antibody 1 x 12µl
    400X Tween-20 1 x 2ml
    AP Development Reagent 1 x 139mg
    Janus Green Stain 1 x 17ml
    Plate Seal 2 units
  • 研究分野
  • 別名
    • MS641
    • MS642
    • MS643
    • MS644

アプリケーション

Our Abpromise guarantee covers the use of ab110217 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
In-Cell ELISA Use at an assay dependent dilution.

MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric) 画像

  • 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.
  • 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.
  • 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.
  • 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.
  • Cells are grown to ~80% confluency in a 96- or 384-well plate, a drug/other treatment is applied to stimulate a cellular response. The cells are then fixed and permeabilized, effectively "freezing" them. Primary antibodies are then added which bind to their intended targets within the mitochondria or other subcellular compartment. After incubation, the unbound primary antibodies are washed away and secondary antibodies are added. These secondaries are conjugated to either IRDyes® or to an enzyme label (HRP or AP) for the colorimetric versions of the assays. Unbound secondaries are washed away, reaction buffer is added for the colorimetric assays, and the signal is read on a suitable instrument for the kit type.
    » In-cell ELISA diagram in PDF format

プロトコール

MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric) (ab110217) 使用論文

This product has been referenced in:
  • El-Sikhry HE  et al. Novel Roles of Epoxyeicosanoids in Regulating Cardiac Mitochondria. PLoS One 11:e0160380 (2016). In-Cell ELISA . Read more (PubMed: 27494529) »
  • Timraz SB  et al. In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering. Exp Cell Res 343:168-76 (2016). ELISA ; Human . Read more (PubMed: 27079869) »

See all 5 Publications for this product

Product Wall

reliable data

Excellent Excellent 5/5 (Ease of Use)
Abreviews
We use HepG2 and H9C2 cells for assay, and 384-well format, the plate wahser can improve the assay window and performance a lot. Two key factors for assay, cell status and wash step.
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Abcam user community

Verified customer

投稿 Mar 27 2017

Dr. Andaleeb Sajid

Excellent Excellent 5/5 (Ease of Use)
Abreviews
I am using this kit for high throughput screening of more than 500 compounds. The kit is highly reproducible and I did not observe any lot to lot variation. I would highly recommend this kit.
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Andaleeb Sajid

Verified customer

投稿 Jul 29 2015


(1) Which human and rat cell lines were used for the testing?
A couple different cell lines were used to generate the data. The cell type used for
Figure 1 were HepG2 cells
Figure 2a. 143B osteosarcoma wild type, 143B Rh0 osteosarco...

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Thank you for your phone call today. Please find the reply from the lab below, and let me know if you have any further questions.

In our experience it is necessary to treat cells for a few passages to observed the effect on mitochondrial prot...

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Looking at your data the results do look pretty good and the kit is performing as expected. Looking over the your questions I would like to emphasis that these kits are developed at different times so there are slight variations in experiment...

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Some cell lines may have low level of SDHA – possibly longer incubations may help.


Though not ideal – the levels of COXI could be normalized to the included cell stain (Janus green).

we suggest to perform a dilute acid wash step: after fixation and before permeabilization, incubate cells for 5 min in 0.5% acetic acid in 1X PBS, followed by a PBS wash. Then proceed to permeabilization and the rest of the protocol. The acetic acid sh...

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Malheureusement, ils utilisent principalement ce kit pour étudier l'inhibition de la réplication ADN mitochondrial. Cependant, ce kit pourrait en théorie être utilisé pour mesurer l'augmentation de l’ADN mitochon...

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"The pH should be 9.8. We can send QC data to the customer if we know the exact lot number (letter followed by 4 digits) written on the label of the tube. We have checked all the buffers made in the last year and without exception they...

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Looking over the data set it appears that the HRP signal has a good signal:background ratio. With regards to the AP signal, it does appear to have high background in control wells. We suggest performing a dilute acid wash step: after fixation...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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