Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911)

The protocol makes specific reference to mitochondria and tissue extracts. When it comes to cells, there is a significant loss of protein in the pellet after extraction as you mentioned on the phone. I would recommend either re-measuring the concentration again after extraction or to load at multiple concentrations (both controls and test samples) to account for potential losses during the extraction procedure. I realize that measuring twice could be very tedious. A way around it is to resuspend the cell pellet in 8 volumes of the buffer supplied = 50uL pellet + 400uL buffer. This should be approximately 5mg/mL, then the detergent can be added which can be followed by a BCA assay.
Below is a link to a sample preparation guide for our Mitosciences ELISA assays and immunocapture activity assays, such as ab109911.
https://www.abcam.com/index.html?pageconfig=resource&rid=15500

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.

We provide the cytochrome c in reduced form for this kit.
I can say the protein is active and also the detergent is mild. So if cytochrome c functions as a dimer then you can anticipate this conformation in the assay solution.
We are unsure whether the molecule will specifically be dimerized or not following addition of the detergent, but you should measure the total concentration using a standard BCA or Bradford assay and use a 5 mg/ml solution with ab109911.

Thank you for your technical enquiry and interest in our products.

I have discussed this with my colleagues in the Lab. This kit (ab109911: Complex IV Rodent Enzyme) measures in a standard spectrophotometer the absorbance of reduced cytochrome c. The peak of absorbance of reduced cytochrome c is at 550 nm. This peak is unfortunately is very narrow and the readings drop quickly after 555 nm. At 570 nm the reading will be almost 0.0.

Apologies!

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for your interest.

The product code number for this item is ab140219. However, it is not published so the datasheet can't get accessed on the public site.

I would advise to place an order via e-mail: mailto:orders@abcam.com and mention that you have been discussing this special order with me.

I have ccd this response to Customer Service so they will be aware of this special order.

If you need any further assistance in the future, please do not hesitate to contact me.

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 contact.

I have discussed your enquiry with my colleagues and can confirm that Reagent C is available for special request. The sale price is USD50.00 or GBP40.00 /unit. We currently have this item in stock.

Could you please confirm if you are still interested in getting this item and how many units you wish to purchase?

I look forward to hearing from you soon.