Using centrifugal filter units to concentrate protein
Posted 28 February 2013 - 05:26 AM
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?
Posted 28 February 2013 - 08:15 AM
I always had an alternate hypothesis....
Posted 28 February 2013 - 12:07 PM
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
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?
Posted 28 February 2013 - 06:36 PM
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.
Posted 01 March 2013 - 01:06 PM
Posted 01 March 2013 - 02:22 PM
Posted 04 March 2013 - 08:05 AM
Posted 17 July 2013 - 12:35 AM
Posted 17 July 2013 - 04:36 AM
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.