Mitochondria Isolation Kit for Tissue (ab110168)

I can confirm our Mitochondria Isolation Kit for Tissue (ab110168) is suitable to use with frozen tissue. If using frozen tissues, the tissue should be flash frozen and stored at least at -80ºC.

ab110168 can be used with 20-30 mg tissues however the mitochondrial yield would be 10-100 ug. Please check the downstream application whether this amount would be suitable.
The homogenization protocol we have provided is for 300-500 mg tissue so you can decrease the volumes of buffer proportionally.

Samples fixed in ethanol are not compatible with ab110168. Ethanol fixes/precipitates proteins, so the samples are no longer suitable for organelle isolation.

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.

For a protease inhibitor to use with the Mitochondria Isolation Kit for Tissue, ab110168, we recommend ab201116.

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. However, we cannot guarantee all other mitochondria activity kits available in Abcam catalog can be used for frozen samples. I recommend reviewing the protocol for other kits that should provide this information. If you have any question about this please do not hesitate to let me know. In general, it is better to freeze mitochondrial fraction rather than to freeze mitochondria lysate/extract.

Ab110168 should work perfectly well with mouse peripheral nerve tissue, provided that the quality of the tissue is good.

This kit is not antibody based and therefore it can work with any species.

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

As we discussed over the phone, I have contacted the lab at Mitosciences to get their input into this query. The mouse brain mitochondrial lysate has been prepared using standard mitochondrial isolation procedures. These procedures separate the mitochondria based on differential centrifugation - mitochondria pellet at 12000g after having removed larger cellular components. As such they are highly enriched for mitochondria but not pure. The quality control of the lysate has been performed using mitochondrial antibody cocktails, please see the image attached. This is true of the kit ab110168 as well.

To make an assessment of purity one would need to look at a variety of non mitochondrial components to determine the level of contamination. So in short if you are looking for guaranteed highly pure mitochondria (for example >90%) we wouldn’t recommend these products.

Essentially very pure mitochondiral fractions are best prepared by taking crude mitos and spinning over a gradient (traditionally percoll, metrizamide sometimes used too). This is not a very scalable procedure so not practical for us to do.