Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online

Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)

Reviews (3)Q&A (34)References (84)
Functional Studies - Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)
  • Functional Studies - Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)
  • Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.

Key features and details

  • Assay type: Enzyme activity (quantitative)
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 6 hr
  • Sample type: Cell culture extracts, Tissue

Overview

  • Product name

    Complex IV Rodent Enzyme Activity Microplate Assay Kit
    See all Complex IV kits

  • Detection method

    Colorimetric

  • Sample type

    Cell culture extracts, Tissue

  • Assay type

    Enzyme activity (quantitative)

  • Assay time

    6h 00m

  • Species reactivity

    Reacts with: Mouse, Rat

  • Product overview

    Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911) is used to determine the activity of cytochrome c oxidase in a mouse sample with speed and simplicity. The COX enzyme is immunocaptured within the wells of the microplate and activity is determined colorimetrically by following the oxidation of reduced cytochrome c by the absorbance change at 550 nm. Included in this kit for performance of the activity assay are buffer, detergent, substrate, and 96-well microplate with monoclonal antibody pre-bound to the wells of the plate, allowing for a stream-lined assay.


    Complex IV assay protocol summary:
    - add samples to wells to capture enzyme and incubate for 3 hrs
    - wash wells
    - add reaction mix
    - analyze with microplate reader in kinetic mode for 120 min


    Buffer, detergent, and microplate should be stored at 4°C, Reagent C (reduced cytochrome c) should be stored at -80°C.

  • Notes

    Range of complex IV / cytochrome c oxidase assay kits

    Biochemical assay - ab239711

    Immunocapture with biochemical assay (plate-based)*** - ab109911 (rodent) (this kit) and ab109909 (human)
    *** Most popular assay format

    Immunocapture with biochemical assay (dipstick) - ab109878 (rodent) and ab109876 (human)

    Immunocapture with biochemical assay and ELISA - ab109910 (human) 

    ELISA - ab179880 (human)

     
    Other 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.

  • Platform

    Microplate reader

Properties

Images

  • Functional Studies - Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)
    Functional Studies - Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)
    Figure 1. To determine the activity in the sample, calculate the slope by using microplate software or by manual calculations using one of the two methods shown. Compare the sample rate with the rate of the control (normal) sample and with the rate of the null (background) to get the relative Complex IV activity. (A)The rate is determined by calculating the gradient of the initial slope over the linear region. (B) The rate is determined by calculating the slope between two points within the linear region.
  • Functional Studies - Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)
    Functional Studies - Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)
    Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. All of our ELISA kits utilize highly validated monoclonal antibodies and proprietary buffers, which are able to capture even very large enzyme complexes in their fully-intact, functionally-active states. Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, substrate is added, and enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed). By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.
  • Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.
    Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.Image courtesy of Yao X et al.PLoS One. 2015; 10(10): e0139416. doi: 10.1371/journal.pone.0139416.

    Mitchondiral fractions from the heart tissue of Rats infected by S. pneumoniae, or given PBS sham control, were subjected to measurements of complex I-V activities. Complex I was measured with ab109721 (top left), Complex II + III were measured using ab109905 (top right), Complex IV was measured using ab109911 (bottom left) and Complex V was measured using ab109714 (bottom right).

    Freshly isolated mitochondrial pellets were resuspened in PBS supplemented with 10% detergent provided in the kits. Protein concentrations of these mitochondrial lysates were estimated and 25 μg (for complex I, IV and V) or 100 μg (for complex II+III) mitochondrial protein was used per reaction. Enzyme activities were measured spectrophotometricly in triplicate and expressed as changes of absorbance per minute per mg protein.

References (84)

Publishing research using ab109911? Please let us know so that we can cite the reference in this datasheet.

ab109911 has been referenced in 84 publications.

  • Zhu Y  et al. miR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1. Oxid Med Cell Longev 2022:3182931 (2022). PubMed: 35126811
  • Han G  et al. Dihuang-Yinzi Alleviates Cognition Deficits via Targeting Energy-Related Metabolism in an Alzheimer Mouse Model as Demonstrated by Integration of Metabolomics and Network Pharmacology. Front Aging Neurosci 14:873929 (2022). PubMed: 35431901
  • Huang L  et al. Increased fatty acid metabolism attenuates cardiac resistance to ß-adrenoceptor activation via mitochondrial reactive oxygen species: A potential mechanism of hypoglycemia-induced myocardial injury in diabetes. Redox Biol 52:102320 (2022). PubMed: 35462320
  • Dong XQ  et al. Implication of a novel truncating mutation in titin as a cause of autosomal dominant left ventricular noncompaction. J Geriatr Cardiol 19:301-314 (2022). PubMed: 35572216
  • Dogan AE  et al. PACT establishes a posttranscriptional brake on mitochondrial biogenesis by promoting the maturation of miR-181c. J Biol Chem 298:102050 (2022). PubMed: 35598827
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

11-20 of 37 Abreviews or Q&A

Question

Hi

The products are:

Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Complex II Enzyme Activity Microplate Assay Kit (ab109908)

Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)



Thanks

Regards

Read More
Answer

Thank you for your enquiry.


I can confirm that In each of these kits we use a non-ionic native detergent at an optimized concentration to maintain OXPHOS complex assembly and activity. This detergent is 1% final N-dodecyl-b-D-maltoside (aka lauryl maltoside). This detergent has been used for many years, particularly in the field of mitochondrial biology and in blue native PAGE separation of OXPHOS complexes.

I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact me.

Read More

Question

ab109721 Complex I Enzyme Activity Microplate Assay Kit.
Which positive controls are appropriate to use with assays.
We have complex 1 and complex 4 enzyme need to know how much to use.
Also, we have been using only some strips, the re-freezing, will this affect the activity?

Read More
Answer

Thank you for your enquiry which has been forwarded to me as my colleague, who you spoke to on the phone, is away from the office today.

We would usually recommend to purchase RHM homogenate (ab110347) as positive control sample. I can recommend to review the online datasheet for further information:

https://www.abcam.com/Rat-heart-tissue-lysate-mitochondrial-extract-ab110347.html (or use the following: https://www.abcam.com/Rat-heart-tissue-lysate-mitochondrial-extract-ab110347.html).

The purified complex I or IV proteins are not provided in the kits because we are not able to find a good source for those proteins.

If you have tested the purified complex I and IV proteins with these kits before and proved they work, by all means you can use them as a control.

I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Read More

Question

What is the shelf-life of the reduced cytochrome C in this kit, stored at -20C? We have a kit ordered in January 2011.

Read More
Answer

We have used the reduced Cytochrome C several years after making it, if it is stored at -80.

The key is to thaw it look and make sure it is homogenous and no clumps or particles, and most important a nice red color. Which, when diluted, gives a nice pink solution. i.e. not brown which would indicate it is oxidized. The initial OD in each microplate well when using the product should be 0.3-0.5 ODs.

I hope this helps. Please let me know if you have any questions.

Read More

Question

Customer kindly contacted us about ab109911 Complex IV Rodent Enzyme Activity Microplate Assay Kit to inquire whether taking measurments at 25C instead of at 30C would be acceptable and so should timepoints be adjusted.

Read More
Answer

Thank you for contacting regardingab109911 Complex IV Rodent Enzyme Activity Microplate Assay Kit.

Reading the plate at 25C should not be a problem and we are not aware of any need to adjust reading time points. We highly recommend that you read all your subsequent plates at the same temperature to ensure consistent data.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

n : 1) si se puede llevar a cabo el protocolo con concentraciones alrededor de 1mg/ml (aunque no sea óptimo). Al final sí que tenemos los 50µg en 200µl, pero más diluidos, claro. He entendido que sí, pero con cuidado.

2) Entonces, mi 2a pregunta es: Qué cantidad de detergente (el lauryl maltoside) deberíamos añadir?? Lo digo porque este apso está pensado para 5mg/ml, con lo que no sé si mejor no tocarlo y no importa demasiado si ponemos más detergente o si, por el contrario, debemos añadir sólo 1/5 de la cantidad de lauryl maltoside.

3) Supongo que el resto del protocolo lo seguimos igual (las mismas horas de incubación/lectura) o sabiendo que tenemos als muestras a 1mg/ml mejor cambiar los tiempos de incubación/lectura?? Cualquier recomendación es bienvenida!!.
Muchas gracias y espero que hasta pronto!!
Saludos,

Read More
Answer

ot
Efectivamente podéis probar a llevar a cabo el protocolo con las concentraciones que indicas, aunque nosotros no lo hemos testado a concentraciones tan bajas, no puedo confirmarte al 100% su idoneidad.

Lo ideal sería optimizar las condiciones de extracción mediante valoración de Lauryl Maltoside (1%, 0.5%, 0.25%) y comprobar qué proceso de extracción proporciona la mejor señal. Aunque siendo que la muestra de la que disponéis es limitada creo que deberíais probar directamente a añadir 1/10 del volumen del detergente para tener una concentración final de 1% de homogeneizado (o fracción de membrana enriquecida) y cargar en la placa el mismo día de la extracción.

No recomiendo diluciones de detergente menores a 0.25% de Lauryl Maltoside, y desde luego no debe reducirse la proporción salvo que se use de un modo sistemático con las muestras control.

El resto del protocolo debe seguirse de acuerdo con las instrucciones indicadas en el protocolo de la datasheet, sin cambiar tiempos de incubación.

Espero haber resuelto tus dudas, en caso contrario, no dudes en contactarme de nuevo.

Read More

Question

Dear ,
Thank you very much for your reply. However, due to the nature of our samples (adipose tissue), we are unable to concentrate the protein/membrane sample over 1-2mg/ml following your previous suggestions.Therefore, since we have no more samples available I was wondering whether we could still save and use them in this ab109911 kit.
- The obvious question is, could we still proceed and not throw away our samples? What is your experience with protein samples concentrated around 1-2mg/ml? I must say that we do get the 50µg of protein needed for the coIV reaction, but rather diluted. Therefore, since not the total amount (50µg of protein) but the concentration of 5mg/ml is the critical step, I assume that the main problem could be in the detergent/next step.
- So, if the answer is 'ok, you could go ahead', how do we go forward? What would be the amount of detergent to be used? I assume that after this step, the rest must be the same (50µg of total protein in 200µl, etc.).
I perfectly understand that since we are not in the ideal range, the results may not be optimal. But, again, I hope that you already have performed some sort of 'dose-response' using this kit and you could have results with lower concentrations (around 1mg/ml).
I would really appreciate your answer and, if possible, the sooner the better.
Thank you so much,
Best,

Read More
Answer

Thank you for contacting us again.

The 5mg/mL concentration is an optimal concentration of tissue homogenate prior to the addition of lauryl maltoside. I’m afraid we don’t have a lot of experience with adipose tissue.

However, I am surprised you don’t have enough sample to concentrate since you mentioned you had 50mg of wet tissue available. Would it be possible to get more tissue and prepare a homogenate with a smaller volume (250uL)? If this is not possible then I would suggest solubilizing the homogenate at no less than 1mg/mL, although this may not give optimal results.

Another alternative you may want to consider is to try the CIV rodent activity dipstick assay, which is more sensitive and requires less protein during sample preparation (25mg of wet tissue).

https://www.abcam.com/Complex-IV-Rodent-Enzyme-Activity-Dipstick-Assay-Kit-ab109878-protocols.html

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

Read More

Question

Hi, I have recently placed an order for the following kits:

Ab109721

Ab109875

Ab110172

Ab109911

Ab109908

Would you be able to let me know if these kits have been successfully used with previously snap-frozen tissue that has been stored at -80C? I am waiting on fresh mouse heart tissue, but am wondering if I could also use these kits to assay tissue I already have stored.

Many thanks.

Best wishes,

Read More
Answer

Thank you for contacting us.
These kits will be OK to use with snap frozen tissues.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

What is the guarantee period for these kits?

Read More
Answer

Thank you for contacting us.

The kits are guaranteed for 6 months if stored as recommended on the datasheet.

Please note there are components in the kits that are supposed to stored at different temperatures e.g. -20 or -80C, so we recommend carefully checking the datasheet and protocol booklet.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

This kit is listed as reacting with mouse and rat. Could you tell me what kind of rat samples were tested?

Read More
Answer

Thank you for your call this morning and for your question.

I've confirmed with my colleagues at MitoSciences that this kit has been used with rat samples, specifically with heart, brain, and liver samples. Brain and heart tend to give a stronger signal.

I will also request that we updated the datasheet to make it more clear that the kit is suitable to use with rat samples.

Please let me know if you have any further questions or if there is anything else that we can do for you.

Read More

Question

Hola de nuevo abcam:
Una usuaria tiene una duda sobre el Ab109911 (kit de actividad complejo IV), requiere saber si lo puede usar para muestras congeladas (en el datasheet no dice). Gracias…

Read More
Answer

Gracias por contactarnos. Este kit puede usarse con muestras procedentes de tejidos congelados o pellets de células congelados. De hecho, para llevar a cabo la homogeneización, es más sencillo de moler al estar congelado. Sin embargo, una vez que el homogeneizado celular sufre el proceso de extracción con el detergente suministrado, no se recomienda congelar las muestras, en su lugar recomendamos realizar el ensayo con extractos frescos. Si necesitas más información acerca de la preparación de muestras en el siguiente enlace: http://www.mitosciences.com/PDF/mitosciences-sample-preparation-guide.pdf Espero que esta información te sea de utilidad, en caso contrario, por favor, no dudes en volver a contactarnos.

Read More

11-20 of 37 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com