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.