Overview

  • Product name
    MitoBiogenesis™ In-Cell ELISA Kit (IR)
    See all MitoBiogenesis kits
  • Detection method
    IR
  • Sample type
    Adherent cells
  • Assay type
    Cell-based (quantitative)
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    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 IRDyes® for detection, and so requires a LI-COR® Odyssey® or Aerius® imaging system. An alternate colorimetric version of this kit is available for use with standard plate readers - MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric) (ab110217/MS643).


    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 IRDye®-labeled Secondary Antibodies. IR imaging and quantitation is performed using a LI-COR® Odyssey® or Aerius® system.


    ab110215 (MS642) 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 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. Optionally, total protein levels can also be measured.


    LI-COR®, Odyssey®, Aerius®, IRDye™ and In-Cell Western™ are registered trademarks or trademarks of LI-COR Biosciences Inc


     


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

  • Notes

    Related products

    Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Tested applications
    Suitable for: In-Cell ELISAmore details
  • Platform
    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab110216 in the following tested applications.

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

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent dilution.

Images

  • 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 (IR). In this example, cells were seeded at 3000 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 3.5 µM.
  • Assay reproducibility demonstrated by experiments conducted on three separate days. HepG2 cells were grown for 7 days in the presence of 10 µM chloramphenicol and then measured in duplicate on 3 different days using the MitoBiogenesis™ In-Cell ELISA Kit. The assay was able to record inhibition of mitochondrial biogenesis with an average intra-day CV of 2.3% and an average day-to-day CV of 1.9%.
  • Visual representation of inhibition of mtDNA-encoded protein synthesis. Visually-imaged levels of mtDNA-encoded COX-I are shown in the green 800 channel while levels of nuclear DNA-encoded SDH-A are shown in the red 700 channel. The merged image shows the relative ratios of COX-I/SDH-A protein expression in each microwell. Wells with normal levels of both proteins exhibit a yellow merged color due to approximately equal red and green signals. In contrast, wells with low levels of COX-I and normal levels of SDH-A exhibit an orange color due to the relative lack of green (COX-I) fluorescence. The specific inhibition of mtDNA-encoded protein synthesis by chloramphenicol is thus easily observed.
  • Quantitative measurement of the COX-I/SDH-A protein expression ratio. At all cell concentrations, a constant 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.
  • Antibody specificity demonstrated by immunocytochemistry. Two-color immunocytochemical labeling of cultured cells with the two MS642 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.
  • 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.
  • 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

Protocols

References

This product has been referenced in:
  • Feng JY  et al. Role of the Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of the Hepatitis C Virus. Antimicrob Agents Chemother N/A:N/A (2015). Human . Read more (PubMed: 26596942) »
  • Feng JY  et al. Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C-Nucleoside Monophosphate Prodrug. Antimicrob Agents Chemother 58:1930-42 (2014). In-Cell ELISA ; Human . Read more (PubMed: 24419349) »
See all 5 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

MitoBiogenesis

Excellent Excellent 5/5 (Ease of Use)
Abreviews
This assay kit is easy to use, we can use 96-well plate or 384 well plate. HepG2 cells were seeded at 3000 cells/well in 96 well plates, allowed to grow for 7 days with a serial dilution of chloramphenicol, and then the relative amounts of COX-I, and SDH-A were measured in each well on the Licor Odyssey, Chloramphenicol inhibits mtDNA-encoded COX-I protein synthesis relative to nuclear DNA-encoded SDH-A protein synthesis by 50% at 5.36 µM.

Abcam user community

Verified customer

Submitted Aug 16 2017

Answer

The laboratory uses the Li-Cor Odyssey (TM) imager for detection of ab110216 but the assay is also available in a standard fluorescent format, ab140359, and colorimetric, ab110217.

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Question


I have been using the IR kit (ab110216) and using Chloramphenicol and ddC in HepG2 cells as my control for mitochondrial tox. I have also attempted this with the Colormetric kit (ab110217).

The HepG2 cells were obtained from ATCC and grown in EMEM(ATCC) +10% FBS + 1%P/S

The Chloramphenicol was obtained from Selleck Chem (http://www.selleckchem.com/products/Chloramphenicol(Chloromycetin).html) 100mg diluted in DMSO to 100mM Stock.

ddC was obtained in house at a stock concentration of 100mM DMSO.

Assay Conditions: Complete growth media in a final conc of 0.50%DMSO, 3-fold, 8pt serial dilution with top concentration of 100uM, all compounds tested in triplicate with Janus Green Stain performed after readout and another duplicate plate in parallel readout with 50% Cell Titer Glo.

Seeding Density: Tried 3000c/w and 8000c/w for 7days post drug addition.

Equipment: I use the LICOR Oddysey for IR readout, and Omega



So I have a few questions:

1. On the scientific support website, HepG2 cells were recommended to be seeded at 1500-3000c/w. In pg 13 of the IR kit protocol (attached), cells were seeded at 3000c/w (Su confirmed they were HepG2) for 3 cell doublings (which I assume to be about 5-6 days) and Chloramphenicol was seen to reduce COX-1 relative to SDH-A with a CC50˜3.5uM. On pg 14-15 of the Fluorescent kit protocol (attached), cells were seeded at 6000c/w for 6 days and looks to have about the same CC50 with Chloramphenicol of about 3uM. On pg 17 of the colormetric handbook, cells were seeded at 6000c/w for 3 cell doublings and Chloramphenicol had a CC50 of 8.1uM. My question is what seeding density should I be using for HepG2 if I need to incubate with serially diluted compound for 7 days (one time only, no re-dosing)? The seeding should not vary by kit I presume?

2. Also, I have not seen the same effects on the HepG2 cells with Chloramphenicol treatment after 7 days, when seeding at 3000c/w or 8000c/w (data shown below). What is the source of your drug, final % DMSO used, what was your compound dissolved in and what was your highest treatment?

3. On pg 2 of the biogenesis playbook (attached), the chart shows that human HepG2 cells were treated for 5 population doublings with antibiotics and 40uM Chloramphenicol produced about 70% inhibition of mitobiogenesis at the endpoint. What was the seeding density and endpoint in hours (for 5 population doublings), and what was the source of the Chloramphenicol?

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Answer



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 experimental procedure and conditions aren’t specific to each kit. As for the direct questions asked in their query, see below:



My question is what seeding density should I be using for HepG2 if I need to incubate with serially diluted compound for 7 days (one time only, no re-dosing)? The seeding should not vary by kit I presume?Using HepG2 cells for a 7 day treatment we initially seed cells at 3000 cells per well.
Kits have been developed independently and at different times, therefore conditions aren’t necessarily the same between kits.



What is the source of your drug, final % DMSO used, what was your compound dissolved in and what was your highest treatment?



We used chloramphenicol from Sigma
Final Concentration of DMSO was 0.2%
Chloramphenicol stock used was 10 mM prepared in DMSO
Treatment ranged using a 2-fold serial dilution curve highest concentration was 64 micromolar down to 0.25 micromolar, and an untreated group





What was the seeding density and endpoint in hours (for 5 population doublings), and what was the source of the Chloramphenicol?In our hands we determined population doubling to about 30-33 hours for hepg2 cells.
Chloramphenicol was sourced from sigma

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Answer

Thank you for your reply.

The WB looks great now - with the bright band at around 37 kDa. The band at the higher MW is very faint and seems to be specific to your sample or sample preparation as the mitochondrial control lysate does not have this band.

I also looked at the images you had sent earlier. The blots from 2010 also showed a weak band at the higher MW. If the same samples were used, than that would confirm that this band is indeed related to your samples. The bovine mito samples do not show such band.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your reply.

The lab sent me the following information to your questions:

1) Do you have some reference for rat or mouse data?

We used the kit in house on h9c2 rat cells. Both antibodies are rat and mouse reactive.
There is a reference to the use of product with rodent cells:

Liu Y et al. "Molecular analysis of mitochondrial compromise in rodent cardiomyocytes exposed long term to nucleoside reverse transcriptase inhibitors (NRTIs)." vol:12 pp123-34 year: 2012 PubMedID = 22170576 (Mouse In-Cell ELISA)

2) Can the kit be used with primary cells?

Yes if sufficient (recommended) # cells can be seeded into the wells and fixed.

3) You have “Figure 2b” in the manual which is an immuno-cyto-chemistry. Can we use it on tissue?
This is validation data for the antibodies to show specific binding of the antibodies to mitochondria target.

To do tissue I am not certain, if it is tissue section I would recommend the individual antibodies and IHC procedure: ab14705, ab14715.

Also please note this kit requires IR plate scanner (LiCor Odyssey or a similar instrument).

An alternative to this assay which can be done with a fluorescent microplate reader (ab140359) will be released soon.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your email.

Cow is another tested species.
I am checking with the lab regarding references or data for mouse/rat, as well as if the kit can be used with primary cells or tissue.

I will be in touch when I hear back from them.

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Answer

Oh, I see.

The bovine heart mito lysate was prepared with iso-osmotic buffer, thus without detergent - so that it can be used for various experiments not just WB.

Additional information as per datasheet:
2 mg of bovine heart mitochondria in iso-osmotic buffer. This sample can act as a positive control in immunocapture applications and Blue Native PAGE.

1 mg solubilized mitochondria for Immunocapture experiments. For the Immunocapture of OXPHOS complexes, it is recommended that 180 µl of the mitochondrial membranes supplied be solubilized by the addition of 20 µl of 10% lauryl maltoside. Insoluble material is removed by centrifugation at 72,000g before immunocapture of complexes from the supernatant. For more detail see Abcam's suggested protocols.


Thus, the preparation of the lysate in this way, works to obtain the expected band at 37 kDa. It seems therefore that the use of 8M urea has a different affect on your samples which might cause it to run at the higher MW.

I would suggest - in light of your results - to try lysing your cells either with an iso-osmotic buffer (mechanical lysis) or an SDS-based buffer. That should help with obtaining the band at the expected MW.

I hope this is of help. Please let me know if you have more questions.

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Question
Answer

Thank you for your reply.

The lab let me know in the meantime that they think you have used too much bovine heart mito lysate. Usually, the loading amount for the bovine heart mito lysate should be at least one tenth of the cell lysates. The band shows up at the correct MW, the additional smear above could be due to overloading the well.

Also, the lab agrees that you will get better result if you use SDS-loading buffer instead of 8M urea for denaturing.

Our WB protocol contains the information about the SDS loading buffer:

https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A6

"The standard loading buffer is called 2X Laemmli buffer, first described in Nature, 1970 Aug 15;227(5259):680-5. It can also be made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in a 1:1 ratio with the sample.

Laemmli 2X buffer

4% SDS
10% 2-mercaptoehtanol
20% glycerol
0.004% bromophenol blue
0.125 M Tris HCl

Check the pH and bring it to pH 6.8.

When SDS is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. SDS denatures proteins by “wrapping around” the polypeptide backbone. SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - i.e., the denatured polypeptides become “rods” of negative charge clouds with equal charge or charge densities per unit length.

In denaturing SDS-PAGE separations, therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. SDS grade is of utmost importance: a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size by using ß-mercaptoethanol or dithiothreitol (DTT).

Glycerol is added to the loading buffer to increase the density of the sample to be loaded and hence maintain the sample at the bottom of the well, restricting overflow and uneven gel loading."



The bovine lysate does not show a band around 85-90 kDa, thus indicating that the kit and antibodies work as expected. It might be that the way your samples were prepared differs from how the bovine lysate was prepared, thus resulting in the difference in MW of the Cox-1 bands.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your email.

Was the bovine heart mitochondrial lysate also prepared with 8M urea?

I am not sure if this is what the lab recommends for denaturing. I think regular loading buffer with 1% SDS would be recommended (Laemmli buffer).

I will check with them, but in the meantime please let me know as well.

Thank you!

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Answer

Thank you for contacting us.

The lysate I am sending you free of charge is just that - a lysate (ab110338, Bovine Heart Mitochondrial lysate, https://www.abcam.com/bovine-heart-mitochondrial-lysate-ab110338.html)

You would load it onto your gel like your other samples, and then use the antibodies in the kit - as you previously did to detect the proteins in the membrane after transfer from the gel.

You would treat the bovine heart mito lysate the same way you treat your samples (e.g. add SDS loading buffer, warm up to 37 degree, add DTT freshly).

Please let me know what your results look like.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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