FAQ
When you dialyze a sample there is a physical tendency for the volume to increase, as the buffer outside the membrane enters into the tube until the hydrostatic pressure within equals the pressure outside. This volume gain approaches zero as the tube is filled with sample. For small samples, the volume gain could be quite significant, percentage-wise. Is this dilution acceptable? If not, use one of the Clean-Up Kits, which precipitates proteins, allowing them to be resuspended in any volume the user chooses.
Would dialyze overnight at room temperature cause degradation of my sample?
It's always a possibility, but considering that most users will be rehydrating their IPG strips with sample overnight at room temperature anyway, this additional step shouldn't result in any additional degradation. Furthermore, the dialysis usually occurs against a strongly denaturing mixture (e.g., 8 M urea) which would denature proteases as well.
Still, if this is a concern, dialysis can be performed at 4 °C.
Should I use the 8 kDa cut-off or the 1 kDa cut-off?
For 2-D electrophoresis, you are generally better off using the 8 kDa cut-off. The larger the cut-off, the faster and more complete the dialysis will be. There will be some loss of polypeptides below 8 kDa, but these smaller polypeptides don't show up in standard SDS-PAGE anyway.* Therefore, there will be no effect on the 2-D spot pattern if the 8 kDa cut-off is used rather than the 1 kDa cut-off.
*NOTE: Standard Laemmli SDS-PAGE (which uses Tris-Cl buffer in the gels and Tris-glycine running buffer) cannot resolve polypeptides of MW 14 kD and lower. These small proteins migrate at the dye front. To resolve such small polypeptides, a Tris-Tricine system is commonly employed [Schagger, H. and Von Jagow, G. (1987). Analytical Biochem. 166, 368].
How long should I dialyze my sample?
Because there is so much diversity among samples, it is difficult to give a definitive recommendation. However, overnight dialysis should be recommended to begin with.
Can I perform desalting techniques instead of TCA/acetone precipitation to get rid of endogenous small ionic molecules?
Yes, small ionic contaminants can be removed using the Mini Dialysis Kit. This product 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.