ATP synthase Specific Activity Microplate Assay Kit (ab109716)

The capture and detection antibody used in ab109716ATP synthase Specific Activity Microplate Assay Kit are widely cross-reactive, but not tested in prokaryotes. All tested species are listed on the individual datasheets.


Having discussed this with the laboratory they think this is likely not to work on bacterial samples.  There would of course need to be some significant sample preparation and optimization, specifically preparation of E.coli inner membranes:


The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli.

I am sorry we have no alternatives to suggest on this occasion. 

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.

1. I would like to know if you mean, after disrupting cell clumps by pipetting up and down, to freeze the cells "as is"? An homogenization with Teflon dounce is not necessary?

Yes resuspend cells from cell culture by pipetting and freeze as is. This weakens and break the cell wall and allows removal of soluble (non membrane) proteins making the extraction more efficient at a lower detergent concentration and so not disrupting the delicate interactions in the ATP synthase enzyme.

2. The freezing has to be performed with liquid nitrogen or a "-80C freezing; is enough?

Straight into the freezer is OK no need for nitrogen or gentle freezing

3. Can the process be stopped after protein determination and the extract can be kept at -80C or not: in the protocol is allowed?

Yes

4. Can Bradford method be used for protein determination?

Yes

The detector antibody was raised against the ATP synthase subunit beta, mitochondrial:

http://www.uniprot.org/uniprot/P06576

We do not know the targetted subunit of the capture antibody. Our best immunocapture antibodies bind the native folded proteins so often do not work in Western blot, making it very difficult to identify the subunit. Particularly if the binding site occurs at an interface between subunits such as ATP synthase alpha/beta subunits.

The antibody has been fully characterized for ATP synthase specificity and completeness of isolation in this paper:

http://www.ncbi.nlm.nih.gov/pubmed/12110673

In the case of this product the capture binds the F1 domain – probably alpha and beta.

The detector we do know binds the beta subunit.

My colleague in our MitoSciences lab has sent me the below reply to your questions. If you need any additional information, please let me know and I will be happy to help you further.

Note this assay goes in reverse – ATP hydrolysis. This is because it is does not require a membrane gradient but instead acts in reverse to pump protons.
This orientation is acceptable since like ATP synthesis it is oligomycin sensitive. None of the buffers contain oligomycin or other inhibitors.

The IC50 of activity in this assay is 10 nM when the enzyme is assembled correctly (F1+FO). To obtain 90% inhibition I would recommend 100-1000 nM oligomycin.
If 90% inhibition is not achieved then analysis of assembly of F1FO should be considered.

Thank you for contacting us.

Same process for converting all to molarity per amount of protein loaded into well.
Note these are all per cm and should be converted into path length for microplate which should be about 0.6cm.

Extinction coefficients

ab109908 (complex II enzyme activity),
ε600 = 21 mM-1cm-1

ab109909 (complex IV activity),
ε550 = 21.1 mM-1cm-1

ab109716 (ATP synthase activity)
ε340 = 6.220 mM-1cm-1

ab119692 (citrate synthase activity).
Ε412 = 14.1 mM-1cm-1

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

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Our development scientist recommends the following units: The units for MS443: -According the protocol (change of OD505/min)/OD405 -Alternative method (change of OD505/min)/(change OD405/min) The units for MS543: -According the protocol (change of OD340/min)/(change OD405/min) Data is calculated as the relative specific activity and the experimental sample should be compared to normal or control. It may also be helpful to review the published papers in the 'Specific references' section of our online datasheet. I hope this information is helpful. We are happy to help so feel free to contact me again if you have any further questions.

Both MS443 and MS543 allow determination of specific target activities, defined as ratio of target activity to target quantity. For example, to determine specific activity of COX in a well using MS443, the COX activity (in mOD550/min) divided by COX quantity (in mOD405, after initial absorbance reading subtraction and considering only linear portion of the OD405 vs. time curve). If the COX activity (in mOD550/min) of a sample is divided by total protein loaded into a well (in mg, measured by e.g. Bradford), the total COX activity per mg of total extracted protein is obtained. For this MS441 is sufficient. I hope this is helpful. Please contact me again if you have any further questions.

Thank you for your enquiry. We are pleased to provide information: The best way to present the results is simply to say that the activity of ATP synthase enzyme is decreased.  Since you are not measuring ATP synthesis, saying that ATP synthesis is decreased is not accurate.  ATP can also be generated by glycolysis (which is not measured in this case).  We find that the hydrolysis reaction is affected in the same way as the synthesis reaction by oligomycin.  You can use oligomycin to inhibit the ms543 reaction by adding it to the well (mixed with the reaction buffer) and read in the spectrophotometer in the presence of the compound.  Details of how to do this experiment are laid out in kit MT-0X5.  0.2-1uM will induce complete inhibition of the enzyme.  The IC50 of oligomycin is approximately 40nM.  Biologically, the ATP synthase reverses in the presence of low membrane potential in an effort to compensate for it.  I would recommend to read the papers of John E. Walker in regards to his work about the rotary mechanisms of the ATPsynthase enzyme to understand more fully how the enzyme works. I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.