Hi everybody,
I'm talking about these
http://tinyurl.com/Amicon-filters
As I use them almost every day, I must re-use them. But since I don't want to lose my sample (obviously:), I always check them first with BSA and when there are proteins in the flow-through, I discard it.
Anyway, is there any way to ensure they last longer than two or three rounds?
At first I was afraid I put too much ethanol into them (I store them in 20% ethanol when not used), so I started keeping them in water, but that seemed to worsen the situation.
My thoughts what could make it go bad are:
- the storage medium - as above, but as the filters sometimes seem to get bad during the usage, probably not
- when I store them, I put the liquid also in the bottom part. Maybe the filter turns bad, when exposed to liquid from both sides? Probably nonsense though.
- when the filters are spinned for too long and there is left only limited amount of the sample, the top of the filter can get dry and turn bad in result. This seems like the most probably cause. In that case, one would have to spin always so that there is still >1.5 ml (in the case of the 50ml falcons) and concentrate more only quickly in the last run.
So what are your thoughts? Any advice?
We store the centrifugal filters in PBS (top and bottom filled). Add sodium azid to the PBS to avoid bacterial contamination for long time storage (more than 1week). I think while using BSA you block the filter additionally. We reactivate the filters by just centrifuging with water. Of course we always use the filter for the same protein. When you are pipetting the concentrate out beware of not disrupting the filter with the tip. Pipette only on the edges of the filter. Pipette the concentrate couple times on the filter before you remove it to avoid blocking of the filter. Some of the filters we are using for months (more than 50 centrifugations). I hope this helps you.
Hi Tomáš, Susanne has good advice about using NaN3 to fend off bacteria growth.
When I reuse filters, I make sure they didn't dry on top and then add 10% glycerol in PBS with 0.01% NaN3 on top.
Thanks Susanne. I'll start using the buffer for storage.
The pipettes could be the problem, especially as we have now those that are marked outside, so it's rather easy to hit the filter.
I'm always extensively washing the filter with water after the BSA or in between samples, so there should be no carry-over.
Always prewet the filters prior to use...
Do more shorter spins and invert each time in between as opposed to long hard spins... they do clog up progressively. Rinse them immediately - don't leave them sitting around to air dry with proteinaceous residue inside...
Avoid using them for buffer exchange - particularly ionic strength - use slide-a-lyzer cassettes instead. They are best for concentration with the same sample matrix!
avoid organics (EtOH/MeOH) always - store in MQ H2O/your buffer + NaN3!
Handle them gently throughout.
Thanks Justin for the answer.
I always wet the filters before using them.
What do you mean by inverting the filters between runs? I always centrifuge only shortly, mainly because of the speed though.
Unfortunately my protein seems not to like dialysis much, so that's not really an option...
Tomas - what I mean is literally up end them (with the lid on still LOL) or gently pipette the retentate in order to prevent local high concentration build up at the bottom of the 'wedge'. Obviously your protein solution is being pulled through the filter due to the centripetal force leading to a progressively higher concentration at the bottom of said wedge. You can see this as a faint yellow band and watch it swirl inside a glass pastuer pipette as you equalise the concentration... All proteins have a cutoff point for solubility and I feel with some of my small highly basic proteins that the shorter more frequent spins with equilibration of concentrations throughout the top filter part of the unit leads to higher yields at the end of the process and a less blocked filter that can be reused for the same sample/protein species later if need be. If I use them for buffer exchange I do it in small increments, 25% ionic strength change at a time sometimes with incubation for longer time periods bewteen spins at lower concentrations of salt. For example when I make 13C15N labelled proteins for NMR I have to remove NaCl from 400 to less than 5 mM Salt so I do it very gradually and slowly and maximise my yield that way (those isotopes are expensive!). Have fun!
I see, I do that always before every run. I trie to mix it gently.
I will probably make the spins even shorter, although it's really annoying just to stand at the centrifuge and check it all the time :(
BTW is, it OK, if I'm going to use the filters in few hours, to put only water in the top and let them be?
Hi all - I want to ask if you said that you fill both top and below compartment with PBS + 0.02% NaN3, is it fully filled? and at which temperature do you storage this tube after filling with PBS - at room temperature or 4C? Thanks for your answer.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts