Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)

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The sample extraction is native so care needs to be taken to extract the samples at equivalent starting concentrations to maintain the protein : detergent ratio and maintain enzyme integrity and activity.

Step 3 – in this we recommend adjusting the lysed cell sample protein concentration to approximately 5.5 mg/mL as determined by some form of protein assay (you can take a small % of sample for this – it may need some prior lysis with SDS or other detergent based on the nature of the sample or protein assay conducted).

Step 4 -Once adjusted it is time for the protein extraction using our supplied extraction detergent.

Wewould recommend doing both steps – step 3 adjustment is precautionary – step 4 is essential to normalize different samples loaded into the plate wells,

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

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.

I would not recommend RIPA buffer because it contains SDS a powerful ionic detergent. The MS141 ab109721 Complex I activity assay contains a mild non-ionic detergent lauryl (n-dodecyl) maltoside which preserves Complex I activity.

Please check the sample preparation guide as well:

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

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.

Samples prepared for dipstick can also work in microplate and vice vera. You could reference to the following guide for sample preparation:

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

Yes, you can extract mitos from frozen tissues but be aware that freezen/ thaw cycle would weaken the cell membrane and cause less recovery of intact mitochondria.

A: There will be slightly loss if you extract enzyme from frozen mitos versus fresh mitos due to the degradation. Adding protease inhibitor to your mito prep would minimize the loss.

As long as the tissues are properly frozen, the variant between different time points should be minimum (within weeks). However, multiple freezen/thaw cycle will cause difference. If you plan to extract sample from different days, make sure you make aliquots of your samples. if you plan to compare the enzyme activity from different samples, we highly recommend to prep the samples under the same condition.

I can confirm the the mitochondria prepared with ab110168 can be frozen and stored in -80 C to be compatible for subsequent analysis using ab109721 kit.