At one time or another most scientists working in the ‘biochemical’ sciences will have to dialyze something. Dialysis is a simple technique to exchange a solute’s solution or to separate/purify differently sized molecules. Macromolecules are dialyzed by placing them in size-selective permeable tubing and subsequently equilibrating the sample with large volumes of new buffer: efficient dialysis relies upon appropriate selection of dialysis tubing and effective ‘washing’ that results from large volumes, multiple changes and full equilibration with the new buffer. I have recently been generating many fluorescent analogs of proteins and have used dialysis to remove ammonium sulfate from protein suspensions prior to labeling and to remove unincorporated succinimidyl esters of various fluorophores after labeling. For these steps I have been using Spectra/Por® dialysis tubing from Spectrum Laboratories®.

Spectra/Por® dialysis tubing is made of either regenerated cellulose (RC) or cellulose ester (CE). Cellulose has long been used for dialysis as it is uncharged and does not readily absorb solutes. Further, the selectivity of cellulose membranes is not altered greatly by many chemicals or reasonable pH and temperature ranges. Processed cellulose has crystalline regions and these regions cross-link chains to introduce structural integrity to the cellulose. Depending upon how the cellulose is processed the number of crystalline areas varies and the resultant regions between the cross-links can act like size-selective pores.

 

Many Spectra/Por® membranes are available with molecular weight cut offs (MWCO) from as little as 0.1 kDa all the way up to 300 kDa (CE membranes come in 0.1, 0.5, 1, 10, 100 and 300 kDa MWCOs, basic RC membranes come in 3.5, 6–8 and 12–14 kDa MWCOs and more expensive RC membranes have a range of MWCOs from 1–50 kDa). MWCO values are established by Spectrum® and represent the size at which a solute is 90 % retain during a test period. The small pore sizes available mean that relatively small molecules can easily be processed and I have used dialysis to buffer exchange samples of ~10 kDa with only minimal sample loss. Certain parameters such as pH may however alter MWCOs and so appropriate tubing needs to be established for a particular situation. That said, with my application using simple buffers the MWCOs have proven to be robust. Spectrum suggest that efficient separation can be achieved if the size ratio of the moieties is at least 25, this ratio is easy to achieve for buffer exchange of a protein or removal of unincorporated fluorescent dyes when labeling macromolecules. Equilibration of the membranes is relatively quick, especially for the larger pore sizes that I typically use, for instance, after labeling I can detect unincorporated dye molecules in my equilibration buffer within 2–4 hours during the first wash. This equilibration time means that 4 buffer changes can easily be performed in 24 hours.

 

Spectra/Por® is supplied in an essentially ready-to-go format, no boiling in bicarbonate buffer is needed. Spectra/Por® 1–6 merely need soaking in distilled water for 30 minutes whereas other tubing is supplied pre-hydrated and must be stored at 4 °C. One concern is that these membranes contain trace amounts of heavy metals and sulfur although Spectrum® do also produce specific cleaning solutions and supply Spectra/Por® 7 which comes with minimal contaminants. Membranes can be sterilized and also have two year shelf lives. Finally, Spectra/Por® tubing is supplied in different widths depending upon the scale of dialysis you plan.

 

For me dialysis essentially provides a ‘low maintenance’ way to perform buffer exchange or purify a sample if there is a large size differential. Other methods do exist to perform the same task including ultra-filtration units that are faster and extremely convenient. Dialysis is however, much more cost effective and so if I am not in a hurry for a sample I tend to use it. Further, dialysis does not rely on concentration of a sample and so may be more ‘gentle’ for your sample. The Spectra/Por® range of tubing definitely proves reliable, cost effective and widely applicable.

 

Peter Haggie, Ph.D.

Post-Doctoral Fellow

University of California, San Francisco