Mini Dialysis Kit is designed for the dialysis of small sample volumes with minimal handling and sample loss, offering a simple solution to the handling problems of low-volume dialysis and reducing the pronounced streaking on 2-D gels caused by low-molecular-weight contaminants (Figure 1). The kit contains dialysis tubes, each of which consists of a sample tube with a cap that is fitted with a dialysis membrane. Sample is easily and quantitatively transferred into and out of the tube by pipetting.
Figure 1.Effect of dialysis on 2-D resolution. Sample: E. coli protein extracted with 15 mM NaCl, 8 M urea, 0.5% Pharmalyte pH 3–10, 2% CHAPS. Dialysis: Mini Dialysis Kit 8 kDa, 250 µl, 17 h against 8 M urea. First dimension: Approximately 400 µg E. coli protein, 13-cm Immobiline DryStrip pH 3–10 NL, Ettan IPGphor Isoelectric Focusing System 32 kVh. Second dimension: SDS-PAGE (12.5%), SE 600 vertical electrophoresis system (16 × 16 cm gel). Stain: Colloidal Coomassie™ G-250.
The capped tube is inverted in a stirred beaker containing the solution against which the sample is to be dialyzed. Salts and molecules smaller than the molecular weight cut-off of the dialysis membrane are effectively exchanged through the membrane. Following dialysis, the tube is centrifuged briefly. This forces the entire contents of the dialysis tube into the bottom of the tube, ensuring essentially 100% recovery. The dialyzing cap is replaced with a normal cap for storage of the dialyzed sample (Figure 2). The kit is available with a choice of molecular weight cut-off (either 1 kDa or 8 kDa) and a choice of tubes for sample volumes of either 250 µL or 2 ml. Each kit contains dialysis tubes and associated accessories sufficient for preparing 50 samples.
Figure 2.Schematic of the method used in Mini Dialysis Kit. (a) Cap with dialysis membrane, conical inner sample tube. (b) Introduce sample, screw on cap, and slide tube into float. (c) Invert and dialyze while stirring. (d) Spin briefly to collect sample.
Dialysis times of a few hours to overnight are sufficient to reduce ionic contaminants to a level that does not interfere with first-dimension IEF separation.
Since some detergents, notably Triton X-100 and SDS, form high-molecular-weight micelles at low concentration, they cannot be effectively removed by dialysis. Other techniques, such as sample precipitation with 2-D Clean-Up Kit, must be used to remove these detergents.
Dialysis tubes with caps incorporating a dialysis membrane (tubes for up to 250 µL or 2 mL sample are included in the kit), caps (standard tube caps to seal the tubes following dialysis), floats (floating plastic sponges to suspend the inverted dialysis tubes in the dialysis solution).
Required but not provided
Centrifuge (dependent on size of dialysis tube) capable of low speeds.
Prior to dialysis, samples for native IEF should be solubilized in water while samples for denaturing IEF for 2-D work should be solubilized in a solution containing urea, reductant, and nonionic detergent. See sections 1.6.1 and 1.6.2 for details.
Handle dialysis tubes and caps with gloves.
Dialysis tubes are supplied in 0.05% (w/v) sodium azide and require rinsing before use.
Spinning longer or faster may rupture the membrane.
See section below on dialysis solutions.
A substantial reduction in interfering ions can be achieved by dialyzing 2-D samples against a solution volume at least 40 times the sample volume, for 2 h to overnight.
Dialyze the sample against a solution that has the same concentrations of chaotropes (urea and thiourea) and DTT as the sample. Other more expensive solution components such as CHAPS and carrier ampholytes do not need to be included in the dialysis solution. These components may be added to their required concentrations following dialysis.
Samples for 2-D electrophoresis should be prepared in a solution that will be compatible with first-dimension IEF, including urea, CHAPS, and DTT.
Vivaspin concentrators can also be used for desalting samples