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
    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

  • Notes

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

  • Platform
    Microplate reader

Properties

Images

  • 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.

    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.
  • 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.

Protocols

References

This product has been referenced in:
  • Zhang K & Jiang D RhoA inhibits the hypoxia-induced apoptosis and mitochondrial dysfunction in chondrocytes via positively regulating the CREB phosphorylation. Biosci Rep 37: (2017). Read more (PubMed: 28254846) »
  • Dadson K  et al. The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1a and Pink1. Sci Rep 7:41490 (2017). Read more (PubMed: 28148912) »
See all 33 Publications for this product

Customer reviews and Q&As

1-10 of 37 Abreviews or Q&A

Abreviews
Easy to use, straight forward to standardize with cell lysates. Good specificity with microplate and results reproducible.

Abcam user community

Verified customer

Submitted Aug 31 2018

Abreviews
I used isolated protein from guinea pig placenta to perform the experiment with this kit. There is no activity detected.

Abcam user community

Verified customer

Submitted Feb 07 2017

Answer


1) Yes, the mitochondrial isoaltion kit is suitable for use with all kits listed.

2) Suspend the prepared mitochondria to 5.5 mg/mL as described in the attached document and follow from step 5 (adding detergent to mitochondia) to generate an extract suitable for all kits.

Read More

Answer

The protocol makes specific reference to mitochondria and tissue extracts. When it comes to cells, there is a significant loss of protein in the pellet after extraction as you mentioned on the phone. I would recommend either re-measuring the concentration again after extraction or to load at multiple concentrations (both controls and test samples) to account for potential losses during the extraction procedure. I realize that measuring twice could be very tedious. A way around it is to resuspend the cell pellet in 8 volumes of the buffer supplied = 50uL pellet + 400uL buffer. This should be approximately 5mg/mL, then the detergent can be added which can be followed by a BCA assay.
Below is a link to a sample preparation guide for our Mitosciences ELISA assays and immunocapture activity assays, such as ab109911.
https://www.abcam.com/index.html?pageconfig=resource&rid=15500

Read More

Answer

We provide the cytochrome c in reduced form for this kit.
I can say the protein is active and also the detergent is mild. So if cytochrome c functions as a dimer then you can anticipate this conformation in the assay solution.
We are unsure whether the molecule will specifically be dimerized or not following addition of the detergent, but you should measure the total concentration using a standard BCA or Bradford assay and use a 5 mg/ml solution with ab109911.

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30ug Lung mitochondrion samples from mice in buffer containing 250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.25% protease inhibitor, and 0.5% BSA, pH 7.4 were used for the assay.

Dr. Amit Agarwal

Verified customer

Submitted Jul 24 2013

Answer

Thank you for your technical enquiry and interest in our products.

I have discussed this with my colleagues in the Lab. This kit (ab109911: Complex IV Rodent Enzyme) measures in a standard spectrophotometer the absorbance of reduced cytochrome c. The peak of absorbance of reduced cytochrome c is at 550 nm. This peak is unfortunately is very narrow and the readings drop quickly after 555 nm. At 570 nm the reading will be almost 0.0.

Apologies!

If you need any further assistance in the future, please do not hesitate to contact me.

Read More

Question
Answer

Thank you for your interest.

The product code number for this item is ab140219. However, it is not published so the datasheet can't get accessed on the public site.

I would advise to place an order via e-mail: mailto:orders@abcam.com and mention that you have been discussing this special order with me.

I have ccd this response to Customer Service so they will be aware of this special order.

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for contacting us and your interest in our products. I am sorry for the delay in getting back to you.

The three kits mentioned (ab109721, ab109911 and ab109908) use 10% lauryl maltoside detergent to prepare the sample at 5 mg/mL. Therefore this is interchangeable between all three kits.

In regards to your second question, we find simply adding the buffer to the lipid mixture is enough. There should not be a need for mixing.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

Read More

Answer

Thank you for your contact.

I have discussed your enquiry with my colleagues and can confirm that Reagent C is available for special request. The sale price is USD50.00 or GBP40.00 /unit. We currently have this item in stock.

Could you please confirm if you are still interested in getting this item and how many units you wish to purchase?

I look forward to hearing from you soon.

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1-10 of 37 Abreviews or Q&A

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

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