Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)

製品の概要

  • 製品名
    Complex I Enzyme Activity Microplate Assay Kit (Colorimetric)
    Complex I キット 製品一覧
  • 検出方法
    Colorimetric
  • サンプルの種類
    Cell culture extracts, Tissue
  • アッセイタイプ
    Enzyme activity
  • 全工程の試験時間
    3h 30m
  • 種交差性
    交差種: Mouse, Rat, Cow, Human
  • 製品の概要

    Complex I Enzyme Activity Assay Kit (ab109721) is a kit designed for the analysis of mitochondrial OXPHOS Complex I enzyme activity from human, rat, mouse and bovine cell and tissue extracts.
    Capture antibodies specific for Complex I are pre-coated in the microplate wells. Samples are added to the microplate wells which have been precoated with a specific capture antibody. After the target has been immobilized in the well, Complex I activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. 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.


    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.


    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • 特記事項

    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

    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.

  • 試験プラットフォーム
    Microplate reader

製品の特性

画像

  • ab109721 measures Complex I activity in human cultured cells within the recommended ranges given in the protocol. Example of Complex I activity measured in Hep2 cells is showed. (Note that these ranges depend on mitochondria preparation quality).

  • 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. Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).

  • Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).

  • (A) ab109721  was used to measure Complex I activity in normal and Rho0 human fibroblast whole cell lysates at 1mg/mL. Rho0 cells are cells in which the mitochondrial DNA has been removed and therefore essential Complex I proteins are not expressed. As shown, the rho0 cells showed no/little complex I activity.

    (B) In a similar analysis, 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 in samples loaded at 0.5 mg/mL whole cell lysates.

  • Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)

プロトコール

参考文献

This product has been referenced in:
  • Zhang XL  et al. RNAi-mediated knockdown of DJ-1 leads to mitochondrial dysfunction via Akt/GSK-3ß and JNK signaling pathways in dopaminergic neuron-like cells. Brain Res Bull N/A:N/A (2019). Read more (PubMed: 30634017) »
  • Lee SY & Long F Notch signaling suppresses glucose metabolism in mesenchymal progenitors to restrict osteoblast differentiation. J Clin Invest 128:5573-5586 (2018). Read more (PubMed: 30284985) »
See all 71 Publications for this product

レビューと Q&A

1-10 of 86 Abreviews or Q&A

Question
Answer

Thank you for contacting us.

The sample extraction is native so care needs to be taken to extract the samples at equivalent starting concentrations to maintain the protein : detergent ratio and maintain enzyme integrity and activity.

Step 3 – in this we recommend adjusting the lysed cell sample protein concentration to approximately 5.5 mg/mL as determined by some form of protein assay (you can take a small % of sample for this – it may need some prior lysis with SDS or other detergent based on the nature of the sample or protein assay conducted).

Step 4 -Once adjusted it is time for the protein extraction using our supplied extraction detergent.

Wewould recommend doing both steps – step 3 adjustment is precautionary – step 4 is essential to normalize different samples loaded into the plate wells,

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

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

Complex I activity in HCT116 cells

Excellent Good 4/5 (Ease of Use)
Abreviews
HCT116 cells were cultivated in standard DMEM medium for 48 hrs. Cell preparation was performed as described in the protocol. Protein concentration in each sample was quantified and adjusted by Bradford assay.
We looked at complex I activity dependent on the protein amount applied to the plate (fig.) thus we would suggest to use protein amounts around 200 µg for this assay.
The kit is easy to use but takes a long time to perform. The results seem to be reliable.

Mr. Christian Marx

Verified customer

投稿 Dec 12 2013

Question
Answer

I would not recommend RIPA buffer because it contains SDS a powerful ionic detergent. The MS141 ab109721 Complex I activity assay contains a mild non-ionic detergent lauryl (n-dodecyl) maltoside which preserves Complex I activity.

Please check the sample preparation guide as well:

http://www.mitosciences.com/PDF/mitosciences-sample-preparation-guide.pdf

Read More

Question
Answer

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 blood with good results.



The caveat is that whole blood must be fresh or must be frozen viable (protocols can be found in the literature). Whole blood should not be loaded in terms of mg of protein as most of the protein present in whole blood is albumin, so using the loading guidelines for tissues to load whole blood will result in loading well below the detection level (OXPHOS proteins are present in leukocytes and platelets only). My suggestion for the customer (if they want to use the quantity dipstick kits) would be to mix 300uL of blood with 900uL of extraction buffer, follow the protocol in regards to the rest of the sample preparation (ice incubation, centrifugation, keeping the supernatant and discarding the pellet). I would advise to the customer to add protease inhibitors to the extraction buffer to prevent degradation of proteins after extraction as granulocytes from blood have high levels of proteases, which will be released once the detergent is added.



For loading on the dipsticks, the blocking buffer should be mixed with 50uL – 100uL of extracted blood so that the final concentration of blocking is 1X. This mixture can then be used to resuspend the gold conjugated antibody dried at the bottom of the well. Once the gold is resuspended the dipstick can be added to the well and allowed to wick the protein fully. Results will not be perfectly accurate (as loading cannot be done in terms of mg of protein), but will be more qualitative in terms of percent of signal from control mean or relative units. This approach will therefore require you to set up a control sample, which should be a pooled sample from several normal donors (10 – 20 donors would be ideal). From our experience, whole blood will not be as accurate as measuring isolated PBMCs only. But it will allow you to have a rough estimate of the trend.

The advantage of isolating PBMCs is that they could run both activity and quantity dipsticks/microplates by following the same guidelines of cultured cells.

Read More

Answer

Samples prepared for dipstick can also work in microplate and vice vera. You could reference to the following guide for sample preparation:

http://www.mitosciences.com/PDF/mitosciences-sample-preparation-guide.pdf

Read More
Abreviews
Very easy to follow instructions and to standardize using cell lysates. Good specificity with microplane reader.

Abcam user community

Verified customer

投稿 Feb 28 2018

Abreviews
I used isolated protein from guinea pig placenta to perform the experiment with this kit. Complex I enzyme activity can be detected.

Abcam user community

Verified customer

投稿 Feb 07 2017

Answer

Yes, you can extract mitos from frozen tissues but be aware that freezen/ thaw cycle would weaken the cell membrane and cause less recovery of intact mitochondria.

A: There will be slightly loss if you extract enzyme from frozen mitos versus fresh mitos due to the degradation. Adding protease inhibitor to your mito prep would minimize the loss.

As long as the tissues are properly frozen, the variant between different time points should be minimum (within weeks). However, multiple freezen/thaw cycle will cause difference. If you plan to extract sample from different days, make sure you make aliquots of your samples. if you plan to compare the enzyme activity from different samples, we highly recommend to prep the samples under the same condition.

Read More

Answer

I can confirm the the mitochondria prepared with ab110168 can be frozen and stored in -80 C to be compatible for subsequent analysis using ab109721 kit.

Read More

1-10 of 86 Abreviews or Q&A

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

登録