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[ccp4bb]: Summary: Filters for DLS measurements
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Dear collegues,
here is a summary of the answers I received after my question concerning the filter size for
preparing DLS measurements:
Michael Forstner:
I was using 0.2 um spin-filters (I think they came from Eppendorf) when I ran out of the 0.02 um
filters, and the results were alright. Normally, we are using the MicroFilters from Hampton
without loss of protein. I would assume that if you loose protein, it's because the protein sticks
to the membrane, in which case centrifugation might be a better way to get rid of aggregates in
the first place.
David Sanders:
I have used 0.2 micron filters for DLS with no apparent problems.One of the original papers
describing this method (Methods in Enzymology Vol. 276 p.157 by Ferre-D'Amare and Burley)
use 200 Angstrom pore size ...
Also, you should probably check your filtered sample another way to make sure that your
protein is being trapped and nothing weirder is going on. Run a gel orUV-Vis spectra ??
Clare Stevenson:
I regularly use a 0.1um filter to filter protein prior to DLS. I use the centrifugal filter from Millipore
as this has no dead volume it doesn't waste your precious protein.
Richard Gillilan:
I've found the smaller size filters (0.02) more difficult to use reliably. They seem to break or leak
easily under modest pressure. In some cases, however, they seem to be necessary. I always
try a larger filter first. With some practice you can recover most of the protein only slightly
diluted and filter again with a finer mesh if need be.
Jeroen Mesters:
I normally start with 0.2 micron filters and work my way down if needed. As you indicated,
sometimes you catch away all the protein which is indicative of lost of small aggregates. You
will not be able to make good measurements from these whether you filter or not. You are wrong
however to assume that aggregates and crystal gowth are incompatible!
Paul Hubbard:
The folks at Prot.-Sol. say you can sometimes get away without filtering if you spin the sample
first.
Stephen McMahon:
We never filter our samples prior to DLS. Instead we centrifuge them in a benchtop centrifuge at
maximum speed (the same as we would treat any sample prior to crystallisation). We were
actually shown this by the Protein Solutions rep who visited our lab. We have had no problem
analysing our samples after treating them this way.
Pierre Rizkallah:
The golden question here is the size of your protein. If it is too big to go through 0.02 micron
filters, you won't see a DLS signal. There is no protein in the solution. The protein is in the filter.
Here I do not mean the unit-cell dimensions or contents of the asymmetric unit, but really truly
monodisperse particle sizes in solution. That's where the answer is. Find out the true
aggregation state of your protein, then you know what size filter cut-off to use.
Thanks a lot for all these hints!
Karsten
----------------------
Dr. Karsten Niefind
Institute of Biochemistry
University of Cologne
Zuelpicher Strasse 47
D-50674 Cologne
tel.: +49/221/470-6444 (Secretary: +49/221/470-6440)
fax: +49/221/470-5092
E-mail: Karsten.Niefind@uni-koeln.de
Internet: http://www.uni-koeln.de/math-nat-fak/biochemie/ds/ma/kni/inhalt_englisch.htm