We are struggeling with upconcentrating several DNA-binding proteins in our lab.

We have been using Millipore`s Amicon Ultra and Centriprep Centrifugal Filters with 10K cut-off for both. In some cases we loose up to 80% of the protein to the filter, that is, more than kind off normal loss. Whether it is systematic for DNA-binding proteins or not, we are not sure, but the same problem was happening recently for three protein in house.

Any suggestions to optimization, or change of filters would be greatly appreciated.

Best regards,

Gro
Norstruct 

Hi Gro,

We use concentrators from Vivaspin.  I am not sure what the filter material is, it may be the same as from Millipore, but maybe it is worth a try.

best,

peggy

Hi Gro,

you either have a protein or  a filter problem. Do you have evidence that your proteins are folded/in good shape/not agregated before you do the concentration? If not that might be a protein problem.

We purified several hundred Ciona DNA BD proteins with no major issue in concentration/purification (see Vincentelli et al 2011 Methods) but we noticed during the optimization phase of purification/storage trials that depending on the storage buffer/conditions (temperature/protein concentration) some proteins were sometimes slowly agregating with time (as seen on GF) and where more difficult to work on (that would probably happen whatever protein family you work on when you start to work on hundreds). Usually changing the buffer or making a new batch was the only way to get well behaving proteins. The final procedure (for all DNA BD) was to add glycerol to the Nickel elution fraction (we have these proteins for several years now and they look fine) and diluting the glycerol out (or decreasing its concentration by dessalting) when we needed them.

Renaud