Mitochondria Isolation Kit for Tissue (ab110168)

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Questions for ab110170:

1. Step 3 of the protocol states: Resuspend the cells to 5 mg/ml in Reagent A in a 2-ml microtube. What does the 5 mg/ml refer to?

A: 5mg/ml is the protein concentration that customer determined using a protein quantity assay, e.g. BCA assay.

2. Step 6 of the protocol states: Homogenize the cells with 30 strokes using pestle B. What brand of dounce homogenizing system do you use and/or what is the diameter size of pestle B?

A: We get the Dounce homogenizer from VWR https://us.vwr.com/store/catalog/product.jsp?catalog_number=KT885300-0002



i.e. Large pestle (A) clearance = 0.889–0.127 mm; Small pestle (B) clearance = 0.013–0.064 mm



Questions for ab110168:



1. Step 4 of the protocol states: Add up to 2.0 mL of isolation buffer to the tissue in the homogenizer. Is their a general rule to use for volume of isolation buffer per weight of tissue?

A: adding four volumes of buffer to one volume of tissue sample will usually be a good start.



2. Step 5 of the protocol states: To rupture the cells, perform the number of dounce strokes suggested in table 1. Use pestle A for the initial strokes, then use pestle B for the remaining strokes. What brand of dounce homogenizing system do you use and/or what are the diameter size of pestles A and B?



A: same Answer as above.

Thank you for contacting us.

It is the amount of tissue. e.g. from 3-5 grams of liver tissue ˜2-4mg of mitochondrial yield can be expected.

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 check the Table 1 of protocol for answers;

https://www.abcam.com/ps/products/110/ab110168/documents/ab110168%20Mitochondria%20Isolation%20Kit%20for%20Tissue%20booklet%20%20(Website).pdf

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 enquiry.

I can confirm that this kit can be used for frozen skeletal muscle of mouse. Please follow the "Heart" protocol. It will be key to mince finely the tissue with a razor before using the dounce, as skeletal muscle also contains many collagen fibers that make difficult complete homogenization.

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

Vielen Dank für Ihre Antwort.

Es tut mir sehr leid dass ich in diesem Fall nicht mehr helfen konnte.

Ich habe ein wenig nach vorhandener Literatur gesucht zur Lokalisation von GADPH als auch alpha-Tubulin.

Ich denke Sie haben Recht, dass GADPH mit Motochondrien assoziiert ist. In apototischen Zellen akkumuliert GADPH. Ich schicke Ihnen einen Link zu einer Publikation die ich gefunden habe.

http://www.ncbi.nlm.nih.gov/pubmed/17072346

Für alpha Tubulin hab ich eine Publikation gefunden, die zeigt das Tubulin an Mitochondrien anhaftet.

http://www.ncbi.nlm.nih.gov/pubmed/12087096

Ich würde daher davon ausgehen dass Sie ein Signal mit beiden Antikörpern für Ihr Mitochondrienlysat bekommen.

Ich hoffe diese Information hilft Ihnen weiter bei der Auswertung Ihrer Ergebnisse. Ich wünsche Ihnen weiterhin viel Erfolg mit Ihren Versuchen.

Thank you for contacting us.

Were you referring to ab110168, the Mitochondria Isolation Kit for Tissue? Tje ab11068 is an antibody, so I assume you're talking about this kit.

For ab110170, the cell pellet is frozen in step 2 of the protocol. Typically, we freeze at -80C and the cells need only be frozen for 10min or so (long enough to ensure that they are actually frozen all the way through). The point is to put the cells through a freeze/thaw cycle to enhance subsequent extraction.

For ab110168, I can confirm that the Mitochondria Isolation Kit for Tissue can be used with frozen tissue. We would suggest that the tissue samples should be flash frozen and stored at least at -80°C.

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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Samples may be prepared as purified mitochondria, crudemitochondria. In most cases whole tissue or cell lysates aresuitable but these may require some sample optimization. Mitochondria samples can be prepared bysimple differential centrifugation of homogenized samples. While any crude isolation may be effective, we recommend ourhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-ab110170.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html(with Dounce homogenizer) if using cultured myocytes such as HL-1 cells.If using heart tissues we would recommendhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.htmlorhttps://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html(with Dounce homogenizer).



This kit was optimized to extract complex I into its native form (with all 47 subunits assembled in to a large complex). 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.Specifically for this product - the extraction is performed by adding 1/10 volume of the 10X Detergent to samples at 5.5mg/mL



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 contacting us and your interest in our products.

I can confirm that the Mitochondria Isolation Kit for Tissue (ab110168) can be used with frozen tissue. We would suggest that the liver tissue samples should be flash frozen and stored at least at -80°C.

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

Thanks for your enquiry.

We have two kits that are designed to isolate mitochondria:

1. ab110168 - Mitochondrial isolation kit for tissues
2. ab110171 - Mitochondrial isolation kit for cultured cells

You can check the Protocol booklet links on the online product datasheets for protocol specifics. In general, for both kits, a nuclear pellet is produced by the first spin and a mitochondrial pellet is produced during the second spin.

Thank you for contacting us.

I can confirm that the kit can be used to isolate the mitochondria from human tissues as well.

Please follow the protocol as given in the protocol booklet; https://www.abcam.com/ps/products/110/ab110168/documents/ab110168%20Mitochondria%20Isolation%20Kit%20for%20Tissue%20booklet%20%20(Website).pdf.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews