Using centrifugal filter units to concentrate protein - (Feb/28/2013 )

Has anyone used centrifugal filter systems such as the Amicon Filters by Millipore? e.g. http://www.millipore.com/catalogue/module/c7715

It doesn't give you a good information on what speeds to use and for how long. Do we just optimise it?
I noticed that solution in the filter would be pulled down even by gravity and a 5 min spin at around 3000rpm would filter out most supernatant while it seems like the manual mentioned a time range between 30-45 min.

Another question is, could you reuse these spin columns?

Help/advice appreciated

If i am right they hav given a fixed speed and varying time depending on the molecular weight cut off. i also just saw the manual from the same amicon 0.5ml centrifugal filter units, which we hav in our lab they do say the speed and time.

I have found that slower/longer spins are best, to prevent loss. I usually spin about 1K for 20-30 minute intervals until I achieve the desired final volume. sometimes it takes forever, but after all the other purification steps, I like to be conservative that close to the end

aimikins on Thu Feb 28 20:07:53 2013 said:

I have found that slower/longer spins are best, to prevent loss. I usually spin about 1K for 20-30 minute intervals until I achieve the desired final volume. sometimes it takes forever, but after all the other purification steps, I like to be conservative that close to the end

Under the specifications tab on the webpage it gives these speeds: Swinging bucket rotor: 4,000 x g; Fixed angle rotor (35°): 5,000 x g

I reuse them several times but within the same day and for the same sample. You don't want the membrane to dry out between each use either.

^{How long it will take to concentrate is directly related to the size of your protein and the MWCO of the filter unit. I use the Amicon 0.5, 4, and 15ml concentrators and have never had a problem using them at the manufacturer's recommendation - just do shorter intervals of time then necessary so you're sure you don't over-concentrate. I've never reused amicon units but have with vivacells and rinsed them well and stored them in 20% ethanol in the fridge between uses.}

The Amicon one's actually dry out due to gravity. So its hard to reuse them, hence the question. Maybe I can fill up both ontop (in the filter) and bottom in the tube to full capacity to avoid drying out?

You can fill up both the top and the bottom. I also believe you can add some glycerol to the top solution which can aid in prevention of membranes drying.....

Do you know if it's better to spin this centrifugal filter devices at low temperature (e.g. 4deg.C, double spinning time compared to at room temperature) so that the protein function can be maintained? Or it doesn't matter if it's used at room temperature?

I think it is protein dependent. Much of my work is with antibodies who behave fine at room temp so 4C isn't necessary but with some proteins crudely purified from lysates I've been more concerned and spun @ 4C and it was fine but it takes a long time. I wouldn't do it unless I had reason to believe it was necessary.