製品の概要

  • 製品名
    ATP synthase Enzyme Activity Microplate Assay Kit
    ATP Synthase キット 製品一覧
  • 検出方法
    Colorimetric
  • サンプルの種類
    Cell culture extracts, Tissue, Suspension cells, Tissue Extracts, Cell Lysate, Purified mitochondria
  • アッセイタイプ
    Enzyme activity
  • 全工程の試験時間
    6h 00m
  • 種交差性
    交差種: Rat, Cow, Human
    非交差種: Mouse
  • 製品の概要

    ATP synthase Enzyme Activity Microplate Assay Kit ab109714 is used to determine the activity of ATP synthase (Complex V) in a human or rat sample.


    The ATP synthase enzyme is immunocaptured within the wells of the microplate and the enzyme activity is measured by monitoring the decrease in absorbance at 340 nm. The conversion of ATP to ADP by ATP synthase is coupled to the oxidation reaction of NADH to NAD+ with a reduction in absorbance at 340 nm.


     


     

  • 試験プラットフォーム
    Microplate reader

製品の特性

画像

  • Examples of activity/load relationships for cultured cell lysate, rat liver mitochondria, and bovine heart mitochondria.
  • Functional study using ATP synthase Enzyme Activity Microplate Assay Kit (ab109714).
  • Principle of ATP synthase Enzyme Activity Microplate Assay Kit (ab109714).

プロトコール

参考文献

This product has been referenced in:
  • Merz TM  et al. Mitochondrial function of immune cells in septic shock: A prospective observational cohort study. PLoS One 12:e0178946 (2017). Functional Studies . Read more (PubMed: 28591158) »
  • Sun S  et al. Loss of the novel mitochondrial protein FAM210B promotes metastasis via PDK4-dependent metabolic reprogramming. Cell Death Dis 8:e2870 (2017). Read more (PubMed: 28594398) »
See all 12 Publications for this product

レビューと Q&A

1-10 of 24 Abreviews or Q&A

Abreviews
Clear instructions, easy to standardize, good specificity with microplane readers, reproducible results.

Abcam user community

Verified customer

投稿 Feb 27 2018

Answer

Thank you for your telephone enquiry earlier today.

I contacted the laboratory who confirmed that the 10X detergent included with this kit is 5% Lauryl Maltoside. The closest product we have is 10% Lauryl Maltoside Solution (ab109857). https://www.abcam.com/10-lauryl-maltoside-solution-ab109857.html

You would need to dilute with this with dH2O to 5% before using it in the kit.

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

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Answer

Yes, typically researchers have used citrate synthase activity to normalize the quality of sample preparation. However, it is also well known in the literature that cell lines with defects in the OXPHOS complexes have increase citrate synthase activity, which completely defeats the purpose of using it as a control parameter. An option here would be to measure specific activity with the following kits.

https://www.abcam.com/atp-synthase-specific-activity-microplate-assay-kit-ab109716.html

https://www.abcam.com/complex-iv-human-specific-activity-microplate-assay-kit-ab109910.html

These assays allow determination of enzyme activity per unit of specific protein (complex V and complex IV). This is achieved by measuring the activity on day 1, leaving the activity buffer on the plate overnight and the next day measuring in the same well the levels of enzyme with a standard sandwich ELISA. This will allow you to know whether increase or decrease in activity is due to changes in the levels of the proteins themselves or other potential post-translational issues.

Another option could be using viability assays for which there are many options out there. One is at this link:

https://www.abcam.com/mitochondrial-viability-stain-ab129732.html

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Answer



- No hay ningún problema en realizar el ensayo a 37 ºC. Simplemente irá mas rápido y no podréis comparar los resultados obtenidos con los de la datasheet, o con publicaciones anteriores que pudieran resultaros útiles.La clave a la hora de hacer ensayos de actividad es tener en cuenta únicamente la parte de la recta con más pendiente para el análisis, y que los ensayos comparativos se corran bajo las mismas condiciones.





-
La opción de medir la inhibición con oligomicina en cada ensayo, o no hacerlo, es realmente una decisión del investigador.

Con correr en cada ensayo un solo test aparte para la Oligomicina debe bastar para validar los resultados. No es necesario hacerlo con cada muestra, si que se recomienda sin embargo hacerlo con la muestra control (control con y sin oligomicina).

Si disponéis de numerosas muestras control, podéis hacer un pool de muestras control y medir la oligomicina una sola vez para cada ensayo.

Si utilizáis toda la placa en un solo experimento, duplicados / triplicados de control con y sin el inhibidor deben bastar para toda la placa.

Si por el contrario se usa la placa en 5 experimentos por separado, lo ideal será correr el control con oligomicina una vez por experimento (5 en total).

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Answer



En respuesta a las preguntas formuladas acerca del kit ab109714:

- Una de ellas hace referencia a la temperatura, ¿por qué el ensayo es a 30ºC? ¿Las enzimas que se utilizan no son de mamífero? El ensayo se lleva a cabo a esta temperatura para permitir una cinética enzimática más rápida. Si se prefiere, puede leerse a temperatura ambiente, pero será más lento el proceso de lectura.

- Respecto a los cálculos, en el protocolo he visto que se expresan como cambio de absorbancia por minuto. ¿Podríais decirme como expresarlo en cantidad de substrato consumida por mg de proteína y por minuto? ¿Qué coeficiente de extinción molar debe aplicarse para el ensayo de ATP sintasa?

El kit mide la disminución de NADH, que de acuerdo con la ley de Lambert Beer, presenta una correlación linear con la absorbancia. El coeficiente de extinción del NADH ε 340 = 6220 M-1cm-1.

La concentración, por tanto, puede determinarse por medio de la siguiente fórmula:

CONCENTRACION = ABSORBANCIA / COEFICIENTE DE EXTINCION * LONGITUD QUE ATRAVIESA LA LUZ (cm)

La longitud de la placa corresponde a 0,6 cm.

- Finalmente, para detectar la sensibilidad a oligomicina, ¿qué protocolo debe seguirse? ¿Se debe correr la misma muestra en paralelo con y sin oligomicina; o es una prueba que se hace a posteriori de la determinación? ¿Qué concentración de oligomicina debe usarse?

Concentraciones por debajo de 1uM inhibirán por completo la enzima. Deben correrse en paralelo las muestras con y sin tratamiento. El valor de IC50 para oligomicina ronda el rango bajo nanomolar. Tener en cuenta que la oligomicina no debe tener más de 3 meses de antigüedad y que debe añadirse al buffer de actividad, que se añade después de los fosfolipidos.

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Question

I purchased your ATP Synthase Enzyme Activity Microplate assay Kit on 1/15/13 and have started doing the experiment with the kit in the past week using rat cell line, H9C2 http://atcc.org/Products/All/CRL-1446.aspx for twice but the experiments did not work. I read your protocol several times and could not figure out what I did wrong. I still have most of my plate left and I need to get the experiment to work. Could you help me to trouble shoot? Here is the information about what I did:

- I immediately stored the plate and Tube 1 at 4oC and froze the Detergent Mix and Phospholipids at -20.


- When I did my experiments in last week, I made the Sulotion I by adding 190ml ddH2O to 10ml buffer in TUBE1, thawed Reagent Mix and Phospholipids at RT than put in ice and aliquoted them. From that time, I used aliquoted, 1x froze and thawed Reagent Mix and Phospholipids for each experiment and never refreeze or use the leftover Reagent Mix and Phospholipids. I am not sure the once freeze and thaw will distroy the sulutions.

- I treated H9C2 cells and after treatment I harvested cells and store pellets at -80oC.

- When I did assay, I resuspent frozen cell pellet in small amount (˜100ul) 1xPBS + proteinase inhibitors to complete homogenous. I froze and thawed the homogenate once in liquid nitrogen and RT then spin to get pellet at 16000rpm for 20min. I got quite nice and big pellets.


- I then resuspend the pellets in 4 volumes of Solution I and measured the protein concentration.


- I then adjusted protein concentration to 5.5mg/ml and added 1/10 detergent. After incubation in ice for 30min, I spinned the samples and got supernatants.

- I loaded 50ug of protein / well, incubated at RT for 3 hours, washed 2x with 300ul Solution I, emptied wells, added 40ul/well of Phospholipid, incubated 45 min, added 200ul Reageat Mix, immediately started reading at 340nm for 2 hours at 1min interval at RT.


- The result I got was attached. I don’t see any color on the sample wells. I am not sure if I should see color on the wells. All the reading values are at zero. My understanding is the initial reading of the wells should be high then I look at the decrease of the reading over the time.


- I also mixed 10ug (about 2ul) of isolated mouse mitochondrion extracted as indicated on your protocol from step 1-8 on Sample preparation section on page 9 and 10 with the Phospholipid, and Reageat Mix by following your protocol step 4 to 7 on page 12, to test if the both reagents should work. I got zero reading at 340nm. I am not sure if this should work.

Anyway, I really don’t know what I need to improve. Could you check what I did wrong and give me some suggestions? Do you have positive control which I can use to test the kit?

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Answer



Any color development is not visible to the naked eye but should easily be detectable to the plate reader.

The wavelength looks right 340nm and there should be a sizeable absorbance due to NADH abs at 340nm (we would expect 0.5), looks like the plate reader is set to max 0.4?

The lab suggested to make up the buffer alone and put it into a well to check the starting OD alone and also try another spectrophotometer. Also be 100% certain that the location of strips being read in reader are the same location as the strips in the frame. If the reader is a spectramax you can read the raw data files, otherwise export text file or file to excel.

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Answer

Thank you for contacting us.Please find the replies to your questions below.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.





ab109714:
1. How to check oligomycin sensitivity? Do I need to check it when I am doing the assay each time?

Only if it is of interest as a control



2. Do I have to use fresh cell pellet or isolated mitochondrion? Can I use frozen pellets from -80oC?

Frozen works well



3. When using frozen cell pellets:
a. Is it correct that I use Solu I to resuspend pellet and pipette the cells to homogenate the cells?

Yes
b. When I do freeze and thaw cycle, should do liquid nitrogen then 37oC?

They can be thawed at room temperature, freezing acts to fracture the membranes and remove any soluble proteins

4. Can I freeze the sample at 80oC for later use after step 7 on page10 in the protocol?

Yes



5. Is it critical to measure activity at 30oC? Our machine measures sample at room temperature.

No not critical – only critical to be consistent between experiments



6. Can I purchase the reagents (detergent, reagent mix, Lipid mix) separately from the 96 well plate? If so, what is the price?

No not currently.

ab109721:
1. Could you let me know the best way to lyse cell pellet (from -80oC) and isolated mitochondrial pellet (from -80oC) for the assay?

Suspend the cells or homogenize tissue to approx. 5.5 mg/mL add 1/10 volume of detergent to lyse the cells.

See step 6.4.



2. Could you let me know how to analyze the raw data? Should I just compare the slopes between samples?

Yes exactly compare the slopes between samples.

Your plate reader should be able to record these slopes when used in the kinetic mode (i.e. with time).

If your plate reader is not able to record in this way, record the entire plate after adding substrate then record plate every 5 mins for 30 mins

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Answer

Product code: 109714 Inquiry: Hi Abcam, Do you know if this kit will immunocapture the ATP synthase enzyme from C. elegans? Unlikely unfortunately – this kit was tested positive with human, rat and cow. Mouse was negative. I think it is unlikely to work with C.elegans. Product code: 109715: Inquiry: Hi Abcam, Do you know if this kit will capture ATP synthase from C. elegans? Thanks! This is the antibody for the above kit 109714. C.elegans is untested. Product code: 109907: Inquiry: Hi Abcam, The instructions to this kit says that you need to add the provided phospholipids or else the reaction will not work ("The phospholipids provided in this kit are essential for Complex V activity"). Why is this so? For example, are the phospholipids necessary for the correct folding of complex V? Thanks! Ab109714 and 109907 are similar products (109907 comes with sample and was designed for inhibition studies). Both kits require the addition of lipids. However you might anticipate that in the absence of lipids the enzyme would freely hydrolyze ATP, this does was not found to be the case.

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Answer

Thank you for your response.

You can certainly freeze down the samples if you wish. I have attached a new version of the Protocol which will be updated/downloaded on the on-line product datasheet as well.

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

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Answer

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

I would suggest trypsinizing the cells to detach them from the culture dish/well or using a cell scraper.

Afterwards the cells can be pelleted and washed, then brought up to approx. 5.5 mg/mL in solution 1.

I hope this will be useful for you.

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