[RASMB] dialyzing membranes

[Frank Niesen]

Hello Eray,

We are very happy using the D-tubes from EMD,
http://www.merckbiosciences.co.uk/Products/ProductDisplay.asp?catno=71506&
They are easy to use, and it is (almost) impossible to punch a hole into the membrane and loose your protein.
I usually dialyse a protein sample of higher concentration (~10x) than necessary, to fit into one dialyse tube "midi".
Than I measure the concentration, and then dilute for the samples needed into dialysis buffer (the second of consecutively used 2x1 liters; first one to discard). The dialysis buffer is also used for the references in AUC.

I am not filling the AUC cells using pipette tips. Rather have all samples prepared in Eppendorf tubes (350 ul) and use a syringe with a 1-ml plastic needle (FPLC tubing) to fill it all in, in one go. The reason is that I found that a volume difference can result from problems with getting the last bit of the second go with the P-200 in (because of the air not getting out, pressing liquid through on the side of the tip).

I hope you will find these information useful.
Best regards,

  Frank 

Dr. Frank H. Niesen

Chemical Biology
Structural Genomics Consortium
University of Oxford
Botnar Research Centre
Oxford, OX3 7LD
United Kingdom

frank.niesen at sgc.ox.ac.uk
skype ID: frankniesen 
phone +44 (0)1865 737834
fax      +44 (0)1865 737231
http://www.sgc.ox.ac.uk/ 


>>> "Eray Dalgakiran" <eraydalgakiran at gmail.com> 14/01/2008 10:22 >>>
Hi everyone!

I have a couple of questions about sample preparation.

We are using dialysis tubing membranes but is there a practical method for
dialyzing samples against buffers prior to measurements? What type of
membranes (company, model etc.) should be used when dealing with protein and
polymers which their sizes are below 200nm (200kDa)? And how can we be sure
the samples' concentrations after dialyzing?

What type of tips should we use with Gilson P-200 that are suitable with
centerpiece holes (company, model etc.)?

Many thanks...



Eray Dalgakiran

Department of Bioengineering
Yildiz Technical University
www.bioeng.yildiz.edu.tr