Hoefer IEF100 - Manual (Page 57)

• p51
Appendix B: Reagents and Solutions
Sample Preparation
Samples prepared for 2D should be fully denatured, free of insoluble
material, and low in overall ionic strength.
Some protein samples will readily solubilize while others are more
difficult, requiring additional reagents (such as thiourea, special
detergents, etc.) to encourage solubilization. The common classes of
additives are listed below.
Denaturants
Urea is the most common reagent used in IEF for disrupting the
internal bonding of the protein, allowing it to unfold. Samples are
prepared using urea concentrations from 8
– 9.5 M. In general, the
higher the urea concentration the better a sample can be solubilized.
Urea reaches its saturation point near 10 M at room temperature.
Thiourea is also used to better solubilize some samples. Frequently,
2 M thiourea is combined with 5 – 7 M urea as a reagent for sample
prep and IPG rehydration.
Detergents
Several types of non-ionic or zwitterionic detergents can be used to
solubilize samples (CHAPS, Triton X100, Nonidet NP-40, and
alkylamidosulfobetaine detergents). CHAPS is the most widely used
detergent for 2D electrophoresis. It is stable in solution. Detergents
such as SDS are not compatible with IEF because they bind to the
proteins and mask the proteins’ native charge.
Reductants
Dithiothreitol (DTT) is commonly used to reduce proteins in IEF.
DTT breaks down in solution, so it is normally prepared and added
just before use. Other reductants such as 2-Mercaptoethanol,
Dithioerythritol (DTE) and Tributylphosphine (TBP) can be used.
Other
Carrier ampholytes or IPG buffers can be added to aid in protein
solubility and help prevent protein precipitation during focusing.
Concentrations of 0.5%
– 2% (v/v) are typically used. Carrier ampho-
lytes may interfere with certain labeling experiments. In those cases,
the reagents should then be omitted from the sample extraction step.
Common classes of additives
Denaturants
Unfold the proteins to expose the
internal native charges.
Non Ionic Detergents
Make samples more soluble without
altering the protein charge.
Reductants
Help break internal disulfide bonds to
further unfold the proteins, and help
reduce the negative effects of oxidation
of proteins during rehydration and IEF.
Other
Carrier amphoytes, proteases, DNases,
RNases.