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Using centrifugal filter units to concentrate protein


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#1 science noob

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Posted 28 February 2013 - 05:26 AM

Has anyone used centrifugal filter systems such as the Amicon Filters by Millipore? e.g. http://www.millipore...ue/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 Posted Image

#2 GNANA

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Posted 28 February 2013 - 08:15 AM

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.
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#3 aimikins

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Posted 28 February 2013 - 12:07 PM

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 :)
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#4 science noob

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Posted 28 February 2013 - 03:05 PM

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 Posted Image


1K as in 1K rpm? or g?

Does the amount of time to take to purify a sample directly correlates to the abundance of the protein of interest in the sample?

#5 PhDinAcronyms

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Posted 28 February 2013 - 06:36 PM

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.

#6 Missle

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Posted 01 March 2013 - 01:06 PM

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.

#7 science noob

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Posted 01 March 2013 - 02:22 PM

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?

#8 Missle

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Posted 04 March 2013 - 08:05 AM

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.....

#9 petenl2004

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Posted 17 July 2013 - 12:35 AM

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?

#10 Missle

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Posted 17 July 2013 - 04:36 AM

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.

#11 petenl2004

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Posted 17 July 2013 - 05:29 AM

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.


Thanks Missle. My protein is crudely purified from yeast whole cell lysates. I guess I should spin it at 4degC then.




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