Complex II Enzyme Activity Microplate Assay Kit (ab109908)

Thank you very much for your call today and for your patience while I have been in touch with the lab scientists regarding this enquiry.

If you're using a tissue homogenate or whole mitochondria sample, before extraction with the kit’s detergent (lauryl maltoside)the protein concentration of the sampleshould be 5.5mg/mL.A way of doing this is as follows:

Sacrifice a small portion of the sample (10 – 20uL), solublize it with any ionic detergent of choice and then determine protein concentration in this small aliquot. That number will be representative (taking into account any potential dilutions made) of the protein concentration of the original sample.

If the equivalent concentration was higher than 5.5mg/mL add more buffer accordingly. If the equivalent concentration was lower than 5.5mg/mL centrifuge the whole tissue homogenate or whole mitochondria at high speed. This will generate a pellet which can then be resuspended in a smaller volume.

Once the sample is at the right concentration,you can add the kit’s detergent and follow the instructions



We have a booklet that explains in more detail sample extraction. This booklet can be found in the following link:

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

I hope that this information will be useful, but if please let me know if you have any further questions or need anything else and I'll be happy to help.

Thank you for your inquiry.


The assays can be optimized by cell number, but since all cells are different sizes we use total protein mass.


In general large cells such as fibroblast, HeLa etc are about 0.25 mg/million. So for a normal cell, 10 million would work.

However lymphoblasts are almost always very small cells and would require even more cells.

One solution is to resuspend the cell pellet to 10 volumes with extraction buffer to get a protein concentration of 2-5 mg/mL.

So to get a 200 uL sample you need to start with 20 uL cell pellet + 180 uL lysis buffer.

We would always recommend using a positive control – a human cell line e.g. HeLa or one of our mitochondrial samples (below)

Name
Species
Applications
Amounts
MitoSciences code
Abcam code

https://www.abcam.com/ab110351
Ms
Immunocapture, BNPage
2 mg
MS822
https://www.abcam.com/ab110351

https://www.abcam.com/ab110348
Rat
Immunocapture, BNPage
2 mg
MS819
https://www.abcam.com/ab110348

https://www.abcam.com/ab110338
Cow
Immunocapture, BNPage
2 mg
MS802
https://www.abcam.com/ab110338

https://www.abcam.com/ab110350
Ms
Immunocapture, BNPage
2 mg
MS821
https://www.abcam.com/ab110350

https://www.abcam.com/ab110347
Rat
Immunocapture, BNPage
2 mg
MS818
https://www.abcam.com/ab110347

https://www.abcam.com/ab110349
Ms
Immunocapture, BNPage
2 mg
MS820
https://www.abcam.com/ab110349

https://www.abcam.com/ab110346
Rat
Immunocapture, BNPage
2 mg
MS811
https://www.abcam.com/ab110346


I hope this information helps. Please contact us with any other questions.

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.

According to the literature, malonate inhibits complex II with an IC50 of ˜40 μM (PMCID: PMC2507763). Please let me know if you have any further questions!

Thank you for contacting us.

Yes, you should keep samples on ice, and also add a protease inhibitor to the detergent.

Yes TTFA can be added and should decrease the activity of Complex II.
However TTFA is not as specific/effective as some of the other OXPHOS inhibitors. Malonate (Buffered malonic acid) could also be used.
Acetylation should be preserved. The capture antibody is a mouse antibody and we have been able to see acetylation by adding a rabbit anti-acetyl lysine antibody ab21623 and Goat anti-rabbit HRP to the plate.
https://www.abcam.com/acetyl-Lysine-antibody-ChIP-Grade-ab21623.html


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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Pipette up and down cells several time for complete lysis and then determine the protein concentration using BCA or Bradford method. Once protein concentration is known then please follow rest of the procedure.

20 million of average sized cells per ml will give 5mg/ml of protein.

The DCIP concentration is 2.5mM.

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.

Es werden keinen zusätzlichen Proteinaseinhibitoren für das Kit (ab109908, Complex II Enzyme Activity Microplate Assay Kit) benötigt.

Wir empfehlen während der Aufbereitung der Proben (bevor das Kit benutzt wird; Proben sollten am Ende 5.5mg/ml in PBS sein) Proteinaseinhibitoren zu benutzen.

We use DCPIP as a the detector in this kit. DCPIP is an enzyme-catalyzed oxidation electron acceptor that is blue in its oxidized form and colorless in its reduced form. A peak in the absorbance is observed at 600 nm, however you will still be able to read signal at 620nm.

Question 1 : what is the composition of the incubation buffer? Does it contain a catalyst or a co-factor?

Incubation buffer is 50 mM Tris.HCl pH7.5, 0.015% lauryl maltoside, and contains 1/20 volume of blocking buffer which is a proprietary casein based blocking buffer. No cofactors, or activators are present that enhance the enzyme activity.

Question 2 : can the protocol be used by skipping step 6 ? Can the incubation buffer be replaced by another solution?

For step 6.6 A blocking reagent is in place as a precaution for some samples to prevent significant non specific binding to the plate, but where significant dilution of sample occurs I would be comfortable with eliminating the blocking reagent from incubation buffer or replacing with Blocking solution with 1% BSA in final incubation buffer

Thank you for your inquiry. This antibody was raised against Bovine (cow) complex II. It is cross reactive with human, cow, mouse and rat and is a mouse monoclonal isotype IgG1. It is Western blot negative so we don’t know exactly which subunit it binds to but we know it isolates purified functional complex II. If you have other questions, please contact us.

We haven't specifically tested the complex I or II kits under reducing conditions and cannot guarantee that it will not affect the enzyme activities. With that being said, my colleagues believe that a reducing agent at moderate concentration shouldn't affect the assay capture step significantly, especially if the reduced samples are sufficiently diluted in the wells.

To convert from mOD/min to nmol/min you can use the extinction coefficient for the dye in that kit.
For the complex I kit (ab109721), the dye Epsilon at 450nm = 37 mM-1 cm-1

The height of 200 uL of liquid in each well is approximately 0.6 cm, therefore epsilon is 22.2 mM-1 well-1 for the complex I kit.

For the complex II kit (ab109908), the dye Epsilon at 450nm = 21 mM-1 cm-1, which for 200uL of liquid (0.6 cm) equates to 12.6 mM-1 well-1.

So the change in absorbance per minute in each well divided by 22.2 for complex I or 12.6 for complex II is the mM oxidized per minute.

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.

Thank you for your inquiry and your patience with this reply.

The incubation solution contains a blocking buffer to prevent non-specific binding with the antibody. Since your sample extracts are not very concentrated to start with, we recommend to always run a background control with the buffer used to extract the sample (as you will be loading the sample neat). If you prepared the sample according to the protocol, this background control should be 1X detergent in PBS. Also, we recommend to run the samples not just at the highest concentration but at least at 2 or 3 concentrations (1:2 and 1:4) to ensure that the signal is not saturated (diluted in the same buffer as the background control) and that regardless of loading concentration the same result is obtained after taking into account a small dilution factor. It is important for you to ensure that the assay is performed within the linear range of the assay.

I hope this information helps. Please contact us with any other questions.

Thank you for your enquiry.


I can confirm that In each of these kits we use a non-ionic native detergent at an optimized concentration to maintain OXPHOS complex assembly and activity. This detergent is 1% final N-dodecyl-b-D-maltoside (aka lauryl maltoside). This detergent has been used for many years, particularly in the field of mitochondrial biology and in blue native PAGE separation of OXPHOS complexes.

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

Thank you for contacting us. I am sorry to hear that the full amount of Ubiquinone 2 was not present in this kit and apologize for any inconvenience this has caused. Provided that this purchase has been made within the last six months, I would be very happy to provide a free of charge replacement to you. In order to process this could you please provide the Abcam order confirmation number or the PO and date of purchase. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for your inquiry.
The 10xdetergent of this kit is Lauryl maltoside 10%.
Thank you.

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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Thank you for your reply.

I will be happy to send out a new kit for you to try. I think I have found your original order, but I just want to double check the address before having the new one sent. Was the original placed on **shipped to:

**

If this is the correct information, then please let me know and I will have the new kit shipped out for delivery next week.

I look forward to your reply.

Thank you for your reply.

I just had one further question, you say that you start off with signal, but it does not decrease with time, as it should. I was curious how long you kept measuring for? I only ask, as some samples take a little longer to start producing signal than others.

By the sounds of it though, I think the best way forward is to go ahead and send out a new kit for you. To do this you could provide either the Abcam order number or the PO# used to purchase the kit?

I look forward to your reply.

Thank you for contacting Abcam.

I am sorry about the issues you are having with ab109908, if we cannot resolve the issues you are having then I would be more than happy to send a new kit.

I just have a few questions for you:

1 - What is the catalogue number of the mitochondrial lysate you are using?

2 - What type of samples are you testing?

3 - Is there any signal coming from the kit, or are all wells just giving a '0' reading?

4 - Would it be possible to send a copy of the results that you are getting with this kit, so that I can talk to the lab about this?

I look forward to your reply and helping to resolve this issue?

Thank you for contacting us.

The lab let me know that +4C at either speed would befine for both kits.

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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These solutions are not the same… different buffers and 10-fold different detergent concentrations.

I hope this is helpful. Please contact us again if you have any further questions.

Thank you very much for your call yesterday and for your patience while I'vebeen in touch with the labregarding your enquiry.

We couldn't find a record of the kit being measured at 450 nm or of previous instructions in the protocol to measure at 450 nm. 600 nm is the optimum absorbance for this particular dye (highest absorbance). But these dyes have pretty wide absorbance ranges (shoulders on the curve) so it's likely the decrease in color could be measured at wavelengths other than 600, butwe can't say for sure. At the very least, the next time we measure it we will record the 450nm spectrum in addition to the 600.

I hope that this information will be useful, but if you have any further questions or concerns please let me know and I'll be happy to help.

Thank you for contacting us.

The lab already got back to me:

step 6. Can use 16,000g for a longer time instead of 25'000g for 20 min?
Answer: Yes, same time - 20 min


step 3. Can dilute sample in 1x incubation solution instead of PBS, becuaseneeds to use 250 ug/well for the assay and cannot dilute further after the detergent step (step 4).
Answer: USE PBS, EXTRACT THE SAMPLE WITH DETERGETNT. FINAL CONCENTRATION IS 5 mg/mL ADD 50 uL TO THE WELL, SO TOTAL AMOUNT 250 ug, DON’T USE INCUBATION SOLUTION AT ALL.



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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Thank you for your reply.

I am sorry for the misunderstanding.

The samples preparation guide is not in addition to the kit protocol but an explanation of the protocol. These arethe steps you should take:

1. Resuspend sample – cell culture pellet (ca 50ul)in 450ulPBS.

2. Add 1/10 volume of Detergent to the sample (e.g. if the

total sample volume is 500 μl, add 50 μl of Detergent).

3. Mix immediately and incubate for 30 minutes on ice.

4. Centrifuge at 25,000 g for 20 minutes at 4°C. Collect the

supernatant discard the pellet.

5. Collect supernatant and quantify protein concentration with BCA method. Make sure the protein concentration is 5 mg/ml. Dilute in incubation solution if necessary.

Now continue with the protocol.

I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

I am afraid the components Tube 1, detergent, and microplateofab109909 may be damagedas they are not supposed to be frozen at all.
The components of ab109908seem to be a bit more robust.
Unfortunately, there is no quick test to check the activity of the kits.


I hope this information is still helpful to your customer.

Thank you for contacting us.

The lab let me know the following:

The question is really interesting. Unfortunately without more testing the answer is no.

We use DCPIP (http://en.wikipedia.org/wiki/Dichlorophenolindophenol) as a the detector in this kit which is not fluorescent. We are not aware of any fluorescent redox dyes that would accomplish this function or a way in which we could couple the reaction.

But we can certainly follow up on it. We are not aware of any other kits like this.

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

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Thank you for contacting us.
These kits will be OK to use with snap frozen tissues.
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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Thank you for your call last week and for your patience while I've been in touch with my colleagues at MitoSciences in regards to your questions.

I've heard back from one of the lab scientists, and following are her suggestions:

"Increasing the homogenate protein to 15 – 25mg/mL may help.My suggestion is to test higher homogenate concentration solubilized with 1% lauryl maltoside as well as 0.5% lauryl maltoside and test them on the plate side by side. Note that the detergent that comes with the kit is at 10% (so 1/10 dilution gives 1% detergent).

The standard concentration for solubilizing proteins for an activity assay is 1% lauryl maltoside. In our experience more detergent is not always best (particularly for complex V and PDH). An example is that a membrane enriched fraction of rat liver homogenate gives a higher signal when the sample is at 25mg/mL than when it is at 5mg/mL, similarly this sample gives a more linear activity when it is solubilized at 0.25% lauryl maltoside than at 0.5% or even 1%.Not all the samples behave in the same way for complex V. Whether this experience will help fix the problem in complex II, I don’t know but it is worth a try.

Another thing you could do is to generate a crude enrichment of membrane proteins before addition of detergent.This can be accomplished by centrifuging the homogenate at 25,000 g for 10 minutes at 4C, discarding the supernatant and resuspending the pellet in half of the original volume in the solution buffer of the assay.This can be followed by protein measurement and addition of the assay detergent. After addition of assay detergent the sample will be the supernatant and the pellet can be discarded.

We have seen that this membrane enrichment is crucial for complex V activity assay, and although we have not tested it in complex II assay I predict it may help to increase the signal."

I hope this informaiton will be useful, but if you have any further questions or if there is anything else that we can do for you, please let me know and I'll be happy to help.

Thank you for your call today and for your patience while I've been in touch with my colleagues at MitoSciences regarding your enquiry.

The OD can be read either every 20 seconds or every 1 minute, whichever is easiest for you to do. In our lab, the OD is measured every 20 seconds, but 1 minute intervals will also give plenty of data.

I hope this information will be helpful, and I apologize for the confusing instructions. If you have further questions or need anything else, please don't hesitate to ask.

We have not worked with this kind of tissue yet. We do have a protocol with general principals of cultured cell and tissue preparation I hope will be useful:


http://www.mitosciences.com/PDF/mitosciences-sample-preparation-guide.pdf
I hope thishelps. Please contact us again if you have any further questions.

Thank you for your call today and for your patience while I have been in touch with the lab about these kits.

I can confirm that the 10x blocking buffers are the same in each kit, soit will be fineto interchange them. This buffer is available for purchase separately as ab126587 as well.

I hope this information will be useful, but please let me know if you have further questions or if there is anything else that we can do for you, and I'll be happy to help.

Thank you for your reply. My colleague Cathrin is not in the office today.

Unfortunately, I do also not know if this kit is compatible with a Tecan reader.

I can suggest to ask the manufacturer of the Tecan reader. They should be able to help you after they had a look at the datasheet of protocol of this kit.

I am sorry I could not be of more help and hope this information is nevertheless helpful.

Thank you for contacting us.

I have some good and some bad news for you. the good ones are that it will work in mice, and that the kits may work with Drosophila samples as we as the homology is for both enzymes compared with the human proteinsis over 98%. The bad one is that you will need quite an amount of material to get your experience running, and this may simply not possible with this test species.

These assays are specific to these enzymes and isoforms as demonstrated by mass spec analysis of immunoprecipitates.


Regarding the buffers, adetergent is not included in either final buffer.

And last but not least, unfortunately the lab couldn't tell me if your "Tecan reader" will work with the kits: they don't know it.



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

Thank you for your reply which has been forwarded to me as my colleagueis currently away from the office.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad kit.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

Regrettably, we are not able to provide information regarding contactdetails of other customersas this is confidential.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Thank you for your inquiry.

1.)

Please follow this link to apreparation guidewith some suggestions for sample preparation.

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

I have copied and pasted the relevant section below for you. The trick is to get the sample to approximately 5 mg/mL during extraction.

Why do we do this – well this prevents a huge detergent:enzyme ratio which could destroy the enzyme or conversely having too much protein to detergent and getting a poor extraction efficiency.

Protein Extraction Method II with 10X Lauryl Maltoside:

1. Resuspend and dilute the cell pellet with 8 volumes of PBS (e.g. 50 μL pellet + 400 μL PBS to a total volume of 450 μL). Add protease inhibitors.
2. Add a 10-fold dilution of 10X lauryl maltoside (e.g. add 50 μL to the cell suspension for a total volume of 500 μL).
3. Mix well and incubate on ice for 30 min.
4. Centrifuge at 16,000 x g for 20 min at 4oC.
5. Collect supernatant and discard the pellet.
6. Measure protein concentration with BCA™ Assay or NanoDrop™ Spectrophotometer.

The protein concentration of the extract should now be ≤ 5 mg/mL, depending on extraction efficiency, as measured by BCA™ Assay. To measure protein concentration with a NanoDrop™ Spectrophotometer, follow the manufacturer’s instructions for measuring protein. In the pull-down menu for sample type, select “Other protein (E 1%),” then enter an extinction coefficient (Ext. Coeff, E 1% L/gm-cm) from Table 3 below.

Table 3: Extinction Coefficients for Cell Extracts

HepG2 16.2

HeLa 14.4

HL-60 17.2

It may be possible to freeze the extract at -80oC before running the assay without significant loss of activity.

2.)I agree with your customer, this looks right.

3.)The solution should be incubated at roomtemperature.

I hope this information is helpful and wish your customer good luck with their experiments.

Thank you for your inquiry. Please find the answers to your customer's questions below:

1. What sequence does the complex II antibody bind to?

Unfortunately this is unknown, due to requirement for native binder we immunize with native enzyme so binding site is very hard to determine.

2. What is the optimal method for mitochondrial preparation? I am using a cell line, and have a protocol in the laboratory for mitochondrial fraction enrichment. Is that sufficient?

Most of our protocols have been developed without the need for mitochondrial isolation therefore we quote amounts of tissue or cell lysates. If you wish to use mitochondria our experience is there is only around a 3X enrichment but this will make the sample more active per mg.

And therefore will likely work well .

3. What concentration of mitochondrial concentrate is necessary? The protocol details recommended concentrations for whole cell extract or tissue mitochondria only.

As with cell culture lysates I would recommend 4-250ug/well

4. Could you tell me the components for the reagents? This is to allow me to better understand how the reactions work- buffer, detergent, blocking solution, activity buffer, lipid mix.



Detergent : Lauryl maltoside 10%

Concentrated Wash Buffer: 0.4M KPi pH7.2, 0.3% LM

Activity Buffer: 20 mM KPi, 0.015% LM

Concentrated Succinate: 1M Sodium succinate.hexahydrate

Concentrated DCPIP : 2.5 mM DCPIP

Concentrated Ubiquinone: 13 mM UQ2

Blocking buffer: Casein based proprietary blocking buffer

Lipids : proprietary mix of phosphatidyl choline


5. Is the microplate strips compatable with the Veritas Microplate Luminometer?

No, I do not believe it is. Veritas is a luminometer – it measures light emission only. Required is a standard laboratory absorbance microplate reader.

http://en.wikipedia.org/wiki/Plate_reader

I hope this information is helpful and wish your customer good luck with their research.

Thank you for contacting us.

I have heard back from the lab with the following information:

All these assays require different sample concentrations described in each booklet protocol - but in most cases is approximately 10ug/microplate well.
The good news is that normal whole muscle homogenate contains a good robust mitochondria signal so less is required, muscle samples are described in most if not all protocols.
Another good news is that the same preparation for any of the assays is suitable for all others. I would estimate that if a 1 mg of homogenate can be prepared (0.2mL at 5mg/mL).
This would provide enough material for each of the assays when diluted to the specific concentration in each assay.
We would recommend - if contemplating doing multiple assays - to include a mitochondrial sample as positive control such as ab110338 isolated mitochondria (note this is cow source)



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

Thank you for contacting us. Please find below the answer to your questions. 1. What sequence does the complex II antibody bind to? This antibody was generated by protein immunization and not by peptide immunization and therefore we don’t know the exact epitope it binds 2. What is the optimal method for mitochondrial preparation? I am using a cell line, and have a protocol in the laboratory for mitochondrial fraction enrichment. Is that sufficient? You don’t need to isolate mitochondria from cells to use this assay. However if you want to use the kit with mitochondria preparation from cells, we suggest to use Mitochondria Isolation Kit for Cultured Cells designed to isolate mitochondria from cultured cells: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html 3. What concentration of mitochondrial concentrate is necessary? The protocol details recommended concentrations for whole cell extract or tissue mitochondria only. Optimal loading for samples not specified in the protocol, will have to be determine empirically by the user. 4. Could you tell me the components for the reagents? This is to allow me to better understand how the reactions work- buffer, detergent, blocking solution, activity buffer, lipid mix. The detergent is Lauryl maltoside (which is also available from the catalogue as a standalone product). The buffer is a phosphate based buffer and the activity buffer contains a ubiquinone, succinate and DCPIP (2,6-diclorophenolindophenol). The reaction is as follows: Succinate + ubiquinone (Q) ------- Fumarate + ubiquinol (QH2) ubiquinol (QH2) + DCPIP ------- ubiquinone (Q) + DCPIPH2 5. Is the microplate strips compatible with the Veritas Microplate Luminometer? It is necessary to have a spectrophotometer plate reader capable of measuring absorbance at 600nm. The complex II reaction does not emit light and cannot be read by luminometry. I hope this information is helpful. Please do not hesitate to contact us if you need further advice or information.

Thank you for bringing this to our attention. An easy way of determining whether the kit is working or not is to use one of the positive controls as shown in the kit protocol (HepG2 lysate, Bovine heart mitochondria, Rat heart mitochondria). BHM and RHM are available from the catalogue. Loading at 250ug/well (2.5ug/uL) should yield -3mOD/min https://www.abcam.com/Bovine-Heart-Mitochondrial-lysate-ab110338.html https://www.abcam.com/Rat-Heart-Mitochondrial-lysate-ab110347.html Protease inhibitors do not affect the assay. It is also possible that Lung cells have a very small amount of complex II (the stochiometry of ETC complexes is not 1:1, so if you get activity with complex I it does not mean you should have a similar activity of complex 2 in the cell line). If this is the case, you may need to isolate mitochondria from the cell line. An option to do this is the following kit: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.html" If you have used all the reagents, I will be happy to send another kit, but I do recommend including the controls, given the possibility that the samples are deficient in Complex II compared to Complex I.

Thanks for your call today and letting us know about the problem with the storage instructions on our shipping boxes. As we discussed, we are sending free of charge kits ab109908 and ab110671 on the order ***, which should arrive tomorrow. I apologize for all the trouble and stress with this issue, and I wish you the best of the luck with your future endeavors. Please let me know if you have any questions or if there is anything else that we can do for you.

I have talked to the lab as to whether you can freeze the samples after step 6 and they have told me that it should be ok to freeze the samples down and then use them the next day. You just have to careful and also make sure that you are comparing frozen to frozen samples and not fresh to frozen.

Thank you for your reply. I have been in touch with our colleagues at Mitosciences who have kindly provided the following information: To answer your question directly, The antibody coating the plate supplied with Kit MTOX1 ab109903 does not cross-react with rat samples (it is human/bovine only).  If you would like to  test complex I activity in Rat mitochondria, it would be better to purchase kit ab109121.  Click here (or use the following: https://www.abcam.com/Transglutaminase-2-antibody-EPR2956-ab109121.html). However, the drawback of kit ab109121 is that it is not rotenone sensitive as it is only the dehydrogenase activity of the enzyme and does not follow the complete reaction (dehydrogenase-ubiquinol reductase).  This is explained in detail in the protocol on the datasheet. To answer the complete question, we have had a substantial experience testing mitochondrial toxicity due to antiretrovirals.  It looks to me that you are treating animals with an antiretroviral compound for HIV?  The effect on mitochondria due to antiretroviral therapy varies tremendously depending on the regime used and to my knowledge the most prominent causes of mito toxicity are : (1) inhibition of chain elongation and/or exonuclease activity of the mtDNA polymerase γ, (2) inhibition of thymidine kinases, (3) oxidative stress, (4) impairment of the adenine nucleotide translocase and (5) reduction in mitochondrial membrane potential (Δψm).  The easiest and most economical way of looking at such a wide spread of potential effects in mitochondria is by testing cultured cells exposed to the compound with the following sets of kits: ab113849 ATP luminescent detection kit = check the scientific support tab as well as the protocol for further insight into mitochondrial toxicity testing.  This can test the mitochondrial bioenergetic state of the cell by comparing treatment in glucose vs. galactose based media. Click here (or use the following: https://www.abcam.com/Luminescent-ATP-Detection-Assay-Kit-ab113849.html). ab113850 JC1 or ab113852 TMRE.  Both of these test membrane potential.  The protocol also has insights as to the effect of low membrane potential in the overall mitochondrial metabolism. Click here (or use the following: https://www.abcam.com/JC-1-Mitochondrial-Membrane-Potential-Assay-Kit-ab113850.html). Click here (or use the following: https://www.abcam.com/TMRE-Mitochondrial-Membrane-Potential-Assay-Kit-ab113852.html). ab113851 DCFDA = which measures ROS. Click here (or use the following: https://www.abcam.com/DCFDA--H2DCFDA-Cellular-ROS-Assay-Kit-ab113851.html). ab110217 and ab110216 Mitobiogenesis In-cell ELISA kits (colorimetric or IR) = These will measure the effect of a compound on the protein levels of a mitochondrial DNA encoded protein (typically affected by nucleoside reverse transcriptase inhibitors) in comparison to the protein levels of a nuclear DNA encoded protein (not affected by NRTIs).  Click here (or use the following: https://www.abcam.com/MitoBiogenesistrade-In-Cell-ELISA-Kit-Colorimetric-ab110217.html). Click here (or use the following: https://www.abcam.com/MitoBiogenesistrade-In-Cell-ELISA-Kit-IR-ab110216.html). The kits above mention can be used with any mammalian cell.  The mitobiogenesis kits can be used with human, rat, mouse or bovine cells.  The protocols explain in detail how to set the experiments.  If you haven’t tested the compound with the first basic acute tests on cells, I would recommend to do this.  It will help to narrow down the potential cause of toxicity or at least help have a viable hypothesis of what could be happening in the animal.  If the first four are normal (after acute treatment) I would suggest for to move ahead with the mitobiogenesis kit after treating the cells for at least 5 – 7 passages.  If they have a very good reason to suspect (due to the chemical structure of the compound) that the effect will be only on the mtDNA polymerase, then my suggestion is to go directly to the ab110217 to confirm the hypothesis. Once you have a reasonable hypothesis of the effect of the compound in-vitro, then choosing the test for in-vivo testing will be much easier.  i.e if the retroviral has an important effect on ROS production then they may want to follow up with antibodies that target ROS induced post-translational effects such as anti-nitrotyrosine, ab110282. Click here (or use the following: https://www.abcam.com/3-Nitrotyrosine-antibody-7A12AF6-ab110282.html). If galactose sensitizes the cells to compound toxicity as measured by the ATP luminescent assay, then very likely the compound is affecting the enzyme activity/assembly/levels of one of the complexes of the electron transport chain.  In this instance, then they will need to determine if it is a direct or indirect effect on the enzymes.  The MTOXC kits (5 kits) will test  whether the compound affects directly any of the enzymes using bovine mitochondria (Note that if a compound directly inhibits the transfer of electrons, by in-vitro testing, in bovine mitochondria, it will very likely affect the transfer in of electrons in other mammalian species such as rat).  MTOXC will test activity in the presence of compound (while the assay runs) The following MS kits: Complex I Enzyme Activity Microplate Assay Kit ab109721, Complex II Enzyme Activity Microplate Assay Kit ab109908, Complex IV Rodent Enzyme Activity Microplate Assay Kit ab109911 and ATP synthase Enzyme Activity Microplate Assay Kit ab109714,  all of which are rat reactive) will test indirect effect of the compound in the activity of enzymes (post-translational modifications, assembly defects) in tissues of treated animals.   ab109721 Click here (or use the following: https://www.abcam.com/Complex-I-Enzyme-Activity-Microplate-Assay-Kit-Colorimetric-ab109721.html). ab109908 Click here (or use the following: https://www.abcam.com/Complex-II-Enzyme-Activity-Microplate-Assay-Kit-ab109908.html). ab109911 Click here (or use the following: https://www.abcam.com/Complex-IV-Rodent-Enzyme-Activity-Microplate-Assay-Kit-ab109911.html). ab109714 Click here (or use the following: https://www.abcam.com/ATP-synthase-Enzyme-Activity-Microplate-Assay-Kit-ab109714.html). These MS kits test activity of tissues or cell extracts after they have been exposed to an experimental condition.  For example if you test NRTI compounds with MTOXC, all results will be normal.  However if you test with the MS kits tissues from animals treated with NRTIs for a prolonged period of time, you will see decrease in activity.  You could use the following antibody cocktails: ab110413 MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail  Click here (or use the following: https://www.abcam.com/Total-OXPHOS-Rodent-WB-Antibody-Cocktail-ab110413.html). ab110414 MitoProfile® Membrane Integrity WB Antibody Cocktail  Click here (or use the following: https://www.abcam.com/Membrane-Integrity-WB-Antibody-Cocktail-ab110414.html). If you believe that the structural membrane integrity of the mitochondria (of treated animals) can have an important impact in complexes assembly and protein levels in specific compartments, then these cocktails may be a good choice.  Note that the ATP synthase Enzyme Activity kit ab109714 measures the reverse reactions (ATP hydrolysis) and not ATP synthesis, however the ATP hydrolysis is still oligomycin sensitive.  A decrease in oxygen consumption by the oxygraph could be due to effects in any of the complexes and not just ATPase.  I.e. rotenone affects oxygraph results, but it is a complex I specific inhibitor. So choosing this early one MS541 kit may lead them into a wild goose chase.  You would need to look at mitochondria in a more holistic way and not just as a measure of ATP synthesis.   I hope this information will be helpful to you. if you have any further questions, please do not hesitate to contact us.

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx. As product may become trapped in the caps of our vials, please remember to spin down any vials before opening. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

Thank you for contacting us.

The kits are guaranteed for 6 months if stored as recommended on the datasheet.

Please note there are components in the kits that are supposed to stored at different temperatures e.g. -20 or -80C, so we recommend carefully checking the datasheet and protocol booklet.

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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Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: #####

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

Thank you for your reply. Unfortunately the PO information which you have given me corresponds to Abcam Order #xxxxx for ab110338 Bovine Heart lysate. Could you please provide this information for ab109908 Complex II Enzyme Activity Microplate Assay Kit? I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information. Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for confirming that information.

I have processed the free of charge replacement and you should have it on Monday, hopefully by early afternoon. Your new order number is **.

If you have any issues with the new kit, or if there is anything else I can help you with, then please let me know.

Hope you have great weekend.