7 replies to this topic

[science noob]

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

[GNANA]

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.

[aimikins]

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

[science noob]

aimikins, on 28 February 2013 - 12:07 PM, 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

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?

[PhDinAcronyms]

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.

[Missle]

^{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.}

[science noob]

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?

[Missle]

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.....