The composition of IEF sample solution
CHAOTROPES – allow proteins to unfold and thus expose their hydrophobic cores. The use of thiourea in combination with urea increases the solubility of proteins in IPG. Thiourea is an efficient chaotrope, although it is poorly soluble in water and requires high concentrations of urea for solubility, the optimal conditions being solutions of 2 M thiourea in 5-7 M urea (1).
SURFACTANTS – solubilise the hydrophobic residues that are exposed as a result of denaturation in chaotropes, i.e. prevent aggregation. The anionic detergent SDS is a very effective protein solubilizer, but because it is charged and forms complexes with proteins, it is not recommended to include it in the solution for the IEF (2). The best choice is to use nonionic or zwitterionic detergents. Traditionally, Triton X-100, NP-40 and sugarbased surfactants, such as octyl glucoside, have been used at concentrations between 0.4-4 (1, 3). In recent years the zwitterionic detergent, sulfobetaine CHAPS has become the surfactant of choice and is generally used at 2-5 in 8 M urea (1).
However, these surfactants are not efficient at protein solubilisation in high concentrations of chaotropes and their solubilising power is further minimised in the presence of highly chaotropic thiourea! In contrast, sulfobetaines with long linear alkyl tails such as SB 3-10are more efficient than CHAPS although they suffer from poor solubility in high concentrations of urea. The sulfobetaine SB 3-10 has been used at a concentration of 2, but is not soluble in concentrations of urea greater than 5 M! (1)
Another group of zwitterionic surfactants is of the amidosulfobetaine type with either a linear alkyl tail, such as ASB-14, or a mixed alkyl-aryl tail, such as C8Ø (the number refers to the number of carbon atoms in the tail). Because these surfactants have more polar head groups than sulfobetaines such as SB 3-10 they are soluble in high concentrations of urea, typically 7-8 M, and are used in concentrations of 1-4. When compared to the conventional IEF sample solution containing CHAPS, these surfactants allow the separation and detection of many more membrane proteins (1, 3-5).
REDUCING AGENTS – reduce disulfide bonds and this leads to complete unfolding of many proteins. Reduction is usually achieved with a free-thiol-containing reducing agent such as -mercaptoethanol (5-15) or dithiothreitol (20-100 mM). However, free-thiol-containing reagents are charged, especially at alkaline pH, and thus migrate out of the pH gradient during the IEF, which results in a loss of solubility for some proteins, especially those which are prone to interaction by disulfide bonding, such as the keratins and keratin-associated proteins. The thiol-containing reducing agents could be replaced with an uncharged reducing agent, TBP (2 mM). Phosphines do not contain a thiol and they can not be alkylated, which leads to a simplified IPG equilibration protocol incorporating reduction and alkylation in a single step (1, 3).
CARRIER AMPHOLYTES – (IPG buffer) enhance protein solubility by minimizing protein aggregation due to charge-charge interactions, and are generally used at a concentration of 0.5, although up to 2 can be used if protein solubilisation is a problem (2, 3).
By combaining thiourea, sulfobetaines such as CHAPS, SB 3-10 and ASB-14, and the uncharged reducing agent TBP, it is possible to create a powerful IEF sample solution and thus solubilise proteins which would remain insoluble in conventional IEF sample solutions. The protein extraction could be done by incorporating the enhanced solubilising conditions as the final step of a sequential extraction. For example, the first step is cell/organelle lysis and protein extraction using Tris buffer. The resulting pellet is extracted using conventional IEF sample solution, or this step could be combained with the first one: 8 M urea, 4 CHAPS, 20-100 mM DTT, 40 mM Tris (in case of soluble proteins use the buffer containing low salt: 10-40 mM Tris-HCl, pH 8.0-8.5 or 40-50 mM Hepes, pH 7.45). The final extraction is with 5 M urea, 2 M thiourea, 2 CHAPS, 2 SB 3-10 and 2 mM TBP (for proteins which require strong surfactants for solubility) or with 7 M urea, 2 M thiourea, 4 CHAPS and 2 mM TBP (for proteins which require a high concentration of chaotropes) (1).
It has to be noted that the choice of a detergent for optimal solubilisation depends on the nature of the proteins to be solubilised, and a detergent suitable for homogenization may not be efficient for IEF (3-5). Several alternatives to sulfobetaine detergents have been recently proposed. N-dodecyl- or -D-maltoside was found to be very effective at a concentration of 2. Another alternative is oligoethylene glycol-based detergent, Brij 56 at 2 (4).
Abbreviations:
IEF – isoelectric focusing;
IPG – immobilised pH gradient;
NP-40 – Nonidet P-40;
CHAPS – 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
SB 3-10 – (N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate);
ASB-14 – 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate;
C8Ø – 4-n-octylbenzoylamido-propyl-dimethylammonio sulfobetaine;
TBP – tributyl phosphine;
Brij 56 – decaethylene glycol mono hexadecyl ether.
Literature:
(1) Herbert B., Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis 1999, 20: 660-663.
(2) 2-D Electrophoresis. Using immobilized pH gradients: Principles and methods, Amersham Pharmacia Biotech, No 80-6429-98, Rev A/ 10-98.
(3) Shaw M.M., Riederer B.M., Sample preparation for two-dimensional gel electrophoresis. Proteomics 2003, 3: 1408-1417.
(4) Luche S., Santoni V., Rabilloud Th., Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 2003, 3: 249-253.
(5) Stanley B.A., Neverova I., Brown H.A., Van Eyk J.E., Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium. Proteomics 2003, 3: 815-820.
2004 02 05 – Julius