altering centrifuge procedure

[adihutama]

Dear all,

I hope Im not re-posting this. I ve searched the forum about this, and it seems that no topic has discussed it yet.

Here's how it goes:
I m trying to do microsome isolation that involve ultracentrifuge _at_ 25000 xg for 15 min. The problem is, although we do have an ultracentrifuge, it cant reach 25000 xg. I know this sound ridiculous, the PIC said that the centrifuge is troubled, so we dont dare to use it in any speed higher than 10500 (or 14500, Im not really sure).
and the question is: *tadaa* how can you replace the 25000xg, 15' protocol? I heard that you can have the same effect if you spin it on lower speed for a longer period, is this true? So what speed and for how long should I spin it in order to gain the same effect as 25000xg, 15''??

Thanks,

Best regards to you all

[canalon]

No it does not sound ridiculous, it sounds like your PIC has a good understanding of metal fatigue and physics of high accelerations, or simply knows a bit about the safety of centrifuge. So limiting the speed of the rotor to prevent destruction of instrument and experience is generally seen as a good idea. Of course a new rotor and/or recertification of the centrifuge/rotor by a competent person might allow it to return to original spec, but that is not cheap.

Anyway, yes it is true that longer centrifugation can replace higher speed. There are some inconvenients, mostly if your microsomes do degrade over time... but you might never achieve complete separation of the tiniest fragments of your samples. But considering your protocol, it looks like it should probably be okay.
Now how long? I do not know. You can try empirical tests. Or you can also have a look at the manual of your centrifuge. There are a lot of tables that explains the correct conversions. Or you can do an approximation and just consider that you want to give the same number of g (yes, I know, but it works, roughly) over the centrifugation so 25000x15=10500x X (with X expressed in minutes). Remember this is not completely equivalent, but it is a good start to estimate the minimum time you will need to get separation.

[adihutama]

that is really some answer I need, thanks a million canalon.

By the way, since I will not use some high speed, I assume usual centrifuge with 14000rpm can do it right? Or the use of ultracentrifuge is compulsory?

[canalon]

rpm and G (or rcf) are not the same thing, though they can be converted in one another (either by the centrifuge itself or the details are in the manual of the rotor).
And as far as your centrifuge and rotors are rated for the speed you want to go, there are no problem using it.