I wanna purify RML strain infected brain of mouse for prion Prpsc....does anybody know approximate yield from one brain of terminally ill mouse?
According to the procedure from Wenborn et al. (2015; Scientific Reports; 5:10062; Figure S2, S3) you could expect to obtain about 14 microgram per mouse brain.
Of course, there are many different purification procedures for PrPSc in the literature. Some give you higher yields than that, but the purity may not be as good. Therefore, the answer depends on what kind of procedure you want to do, which itself depends on what kind of down-stream experiments you have in mind.
Holger is, of course, absolutely right. It all depends on the PrPSc purity you need! Already 25 years ago (McKinley et al., J Virol. 1991 Mar;65(3):1340-51) you could routinely obtain about 30 µg of PrPres out of 1 hamster brain (which, with 0.4 g and 1.0 g for mouse and hamster brains, respectively) translates to about 12 µg purest PrPres per mouse brain.
well i want to transfect N2A cell line for detection on calcineurin activity in them....thanks anyway
To infect N2a cells with RML prions you don't need to purify PrPSc. You can use straight brain homogenate for that.
Also makes the control easy, since you can use normal / uninfected brain homogenate for that.
so do you think N2A cell line will behave same to purified prpsc and homogenate at same level as far as calcineurin activity is concerned?
I know very little about calcineurin. But if you want to test for effects PrPSc may have on calcineurin, it may be sufficient to compare the results from normal and infected brain homogenates, at least for a first set of experiments. If you see a difference in calcineurin, it stands to argue that it is because of PrPSc.
Of course, if you see a difference, then you definitely have to repeat the experiment with purified PrPSc. But for a first experiment you can try just with brain homogenate, which is much easier to do.
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