ATP synthase Enzyme Activity Microplate Assay Kit (ab109714)

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.

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

- 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.





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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).

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.

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.

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

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.

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.

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.