I am measuring ACE activity in lung tissue and have homogenised it this way:
Samples (~ 50 mg) homogenized in 300 μl ice-cold buffer, then centrifuged at 16400 g for 10 min at 4°C. Supernatant removed and stored at −80°C. Pellets were homogenized in a further 300 μl buffer and the supernatants removed after centrifugation.
So I have two supernatants. I need to measure the protein content of the tissue and also perform an ACE assay. Should I measure the protein content of both (with BCA protein assay kit) and take the average? And the same for ACE levels?
Hello...
That's up to you and to whatever is your goal with this experiment...
If the second extraction (ressuspension of the pellet) is intended to "wash" de debris and obtain more protein to analyse, then you should just mix both supernatants prior to freezing (they are the same protein extract).
If the second extraction is intended to extract protein from a different cellular compartment (i.e., if your centrifugation is selective or your second buffer is different from the first), then you should treat them as different extracts and not average them.
I deeply believe it is the first one, mostly because if it was the second one, you would't be asking this question :)
There should be no problem in mixing them after thawing.
There is still another consideration: in case they are indeed just 2 rounds of extraction to obtain more protein, you can still quantify them separately. That way, you can decide if the second extraction rendered a nice amount of protein (and that way you wanna use it) or if it is very diluted already (meaning all protein was successfully extracted in the first round, and in that case it is a better option not to mix nor use the second round, which will only weaken whatever signals you'll be testing).
Best wishes
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