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MitoSciences (MS141)

Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Submit an Abreview Q&A (84)Specific References (8)

Overview

  • Product nameComplex I Enzyme Activity Microplate Assay KitSee all Complex I kits ...
  • Detection methodColorimetric
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts, Tissue
  • Assay typeEnzyme activity
  • Assay durationMultiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    A 96-well microplate assay that is used to determine the activity of Complex I in a human, mouse or rat sample. The enzyme is immunocaptured within the wells of the microplate and activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at 450 nm.

     

    Complex I activity is controlled by enzyme amount and by post-translational phosphorylation at key specific regulatory residues. Cellular metabolism governs these two factors. Ultimately, the cell type and growth conditions will affect Complex I activity measurements.

     

    Note: This activity assay measures the diaphorase-type activity of Complex I. This activity is not dependant on the presence of ubiquinone and therefore inhibitors, such as rotenone, which bind at or near the ubiquinone binding site do not inhibit this assay. However, both the activity assay and the quantity assay described here are affected by enzyme assembly deficiencies.

  • Notes

    ab109721 is shipped at 4°C. 20X NADH and 100X Dye are shipped lyophilized. Rehydrate 20X NADH by adding 1.1 mL H2O. Rehydrate 100X Dye by adding 0.25 mL H2O, then vortex each thoroughly until dissolved. After hydration unused amounts of these two materials should be stored at -80°C. When planning multiple experiments, aliquot these reagents to prevent freeze thaw cycles. Store all other components at 4°C

  • Tested applicationsFunctional Studies more details
  • PlatformReagents

Properties

  • Alternative names
      NADH dehydrogenase

Applications

Our Abpromise guarantee covers the use of ab109721 in the following tested applications.

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

Application Notes
Functional Studies FuncS

Complex I Enzyme Activity Microplate Assay Kit images

  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    ab109721 measures Complex I activity in human and rat cultured cells but also in tissues/tissue mitochondria samples within the recommended ranges given in the protocol. (Note that these ranges depend on mitochondria preparation quality.) Examples of Complex I activity measured in different rat and mouse mitochondrial samples are shown in the graphs above.
  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    (A) ab10972 was used to determine the Complex I activity in normal fibroblasts and rho0 fibroblasts (cells in which the mtDNA is removed and hence essential Complex I proteins are not expressed). The rho0 cells showed no/little complex I activity. In a similar analysis, (B) rat cardiomyocytes were grown for 5 days in ± 40 µM chloramphenicol (CAM) to inhibit mitochondrial protein synthesis, Complex I assembly and hence activity was greatly reduced.
  • Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
    Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Protocols

References for Complex I Enzyme Activity Microplate Assay Kit (ab109721)

This product has been referenced in:
  • Kumarasamy S  et al. Construction of two novel reciprocal conplastic rat strains and characterization of cardiac mitochondria. Am J Physiol Heart Circ Physiol 304:H22-32 (2013). Rat . Read more (PubMed: 23125210) »
  • van den Bosch BJ  et al. Defective NDUFA9 as a novel cause of neonatally fatal complex I disease. J Med Genet 49:10-5 (2012). Functional Studies . Read more (PubMed: 22114105) »

See all 8 Publications for this product

Publishing research using ab109721 ? Please let us know so that we can cite the reference in this datasheet

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we want to use mitochondrial complex 1 kit ab109721. we want to use from cell lysate using blood.there is mention the optiomsation of cell lysate but it is not mention how to optimise the cell lysate kindly help us for these .and also let which prot...

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Red blood cells do not contain mitochondria so isolation of peripheral blood mononuclear cells (PBMC) is recommended.
Once the required amount of cells (number or mass) is isolated I would not anticipate any specific problems.
We haven&rsquo...

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Do you have any recommendations for OXPHOS assays made specifically for blood?

My guess is that the levels of OXPHOS proteins in whole blood are below the level of detection for activity assays. The activity assays for the most part require higher loading than the sandwich ELISA assays. We have tested Complex IV Quantity with whole ...

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Protocol states to use lysate at concentration of 5.5mg/ml. However my mitochondrial lysate is at 1.5-2.5mg/ml. And I only have about 20ul total lysate. Is this enough starting material to use this assay kit?

The protocol suggest a sample loading of 100ug/well (with is equivalent to 0.5mg/mL on a 200uL volume loading). According to what the customer says they only have 8uL at a concentration of 5.5mg/mL. After adding the detergent and centrifuging, they would ...

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Protocol states to use lysate at concentration of 5.5mg/ml. However my mitochondrial lysate from mouse skeletal muscle is at 1.5-2.5mg/ml. And I only have about 20ul total lysate. Is this enough starting material to use this assay kit?

Yes just scale the volume of detergent based on total volume. So in this case 1/10th of detergent would indeed be 2.2uL (final volume should be 22uL).

Protocol states to use lysate at concentration of 5.5mg/ml. However my mitochondrial lysate from mouse skeletal muscle is at 1.5-2.5mg/ml. And I only have about 20ul total lysate. Is this enough starting material to use this assay kit?

For approximately half the protein concentration, adjust the final detergent concentration accordingly.

20uL will be 30-50ug. Since this is muscle this amount, while not a lot, should be enough and could probably used in amounts similar to the r...

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Inquiry: Can I freeze the cells to -80°C for the analysis by Complex I Enzyme Activity Microplate Assay Kit (ab109721) ?



I am happy to confirm that the cells can be frozen in a dry pellet.

It is very important that once the extract is made from that frozen pellet, it must be run immediately.

Thank you for your reply.

I just had one more question: is it necessary to isolate mitochondria or can I use total protein?

Hello!

You can use total tissue homogenate or cultured cell lysate.

These will have lower activity per mass of protein of course.

Figures 2 and 3 show activity derived from whole cell lysates in cultured HepG2 (human liver de...

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Both kits Complex I and II are working fine.
However, the same level of activity was measured with incubation buffer only as for the lowest quantity of protein (20µg).
No activity observed with solution 1 only.

Question 1 : what is the co...

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Question 1 : what is the composition of the incubation buffer? Does it contain a catalyst or a co-factor?

Incubation buffer is 50 mM Tris.HCl pH7.5, 0.015% lauryl maltoside, and contains 1/20 volume of blocking buffer which is a proprietary case...

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starting sample is purified mitochondria, is centrifugation after incubation with the detergent, in Step 6.5 (20' at 12000xg) necessary. Won't this pellet mitochondria?

In step 6.4, the mitochondria will be lysed. Step 6.5 is required to pellet insolubles post-lysis. Save the pellet but it should not contain anything of value assuming lysis was successful.

Inquiry: Complex I Enzyme Activity Microplate Assay Kit (ab109721) May use trypsin to detach the cells or it can interfire with the elisa assay?

Thank you for your enquiry and interest in our products.

I can confirm that enzymatic digestion of the cultured cells with trypsin+EDTA does not interfere with this particular assay (ab109721: Complex I Enzyme Activity Microplate Assay Kit) as l...

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