We performed glycopeptide enrichment by lectins and deglycosylation of peptides on MICROCON centrifugal filters (30 kDa, Ultracell PL 30). Checking for the peptide yield by Tricine PAGE, we sometimes observed the filtration of large amount of peptides, which should be retained by the filter. The event is random, it can happen even with blank samples. We performed the filtration at 14.000 xg, room temperature, using buffers compatible with the Ultracell membrane. We are wondering if it is a problem of (an unlucky) lot of filters. Has anyone had a problem with that?
Thank you for any suggestions.
Francesca
How big are these peptides? You are using filters with a 30 kDa cutoff, so small peptides would be expected to pass through them to some extent. To avoid this, use filters with a cutoff below the molecular weight of the peptides.
Thanks for your answer. In our case, we want only deglycosylated peptides to pass through the filter, but we find too many peptides and the lectins. Then, we have specultated that the filter could be broken. I read the datasheet many times, but I haven't found an answer. Maybe, is there a limit in the number of ultrafitration steps for single filter?
If the lectins are passing through the membranes, the membranes are probably broken.
Never touch the membrane surface with anything.
Don't try to reuse the centifugal filters, if that is an option. You can't clean them effectively, and the surfaces are very delicate.
To save money on single-use filters, use the smallest filters that you can.
" We performed the filtration at 14.000 xg, room temperature, using buffers compatible with the Ultracell membrane. ".. here is your problem.. you are not supposed to centrifuge it at so high rpm. it should not exceed 5000*g. and it should be properly balanced.(weight it with the other blank normal centrifuge tubes. ) so even if you are centrifuging blank filtr ,it is breaking.
Jannatul firdous Siddique
Microcon's datasheet indicate that you can centrifuge the 0.5 ml device at 14000*g for 20 minutes. The filter units were balanced correctly.
I'm waiting for an answer also from Millipore technical support.
Thank you for your advice.
Francesca Raimondo I am also using the same membrane and it can barely with stand 8000rpm. I always centrifuge at 4000rpm for 30minutes and I get 40% of the sample in retentate.
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