Pretreatment of samples for 2D PAGE involves solubilization, denaturation and reduction to completely break up the interactions between the proteins (Rabilloud 1996). Although desirable, there is no single method of sample preparation that can be universally applied due to the diverse samples which are analyzed by 2D gel electrophoresis. The ideal sample solubilization procedure for 2D PAGE would result in the disruption of all non-covalently bound protein complexes and aggregates into a solution of individual polypeptides (Herbert et al 1996). However, whatever method of sample preparation is chosen, it is most important to minimize protein modifications which might result in arte-factual spots on the 2D maps. Samples containing urea must not be heated as this may introduce considerable charge heterogeneity due to carbamylation of the proteins by isocyanate formed from the decomposition of urea. Generally speaking, samples should be subjected to as minimum handling as possible and kept cold at all times (Dunn 1993).
Another consideration is, whether proteins from whole cells or from subcellular fractions (e.g. mitochondriae, ribosomes) (Görg et al. 1997; Corthals et al. 1997), or certain solubility fractions (e.g. a total protein extract or protein fraction such as water-soluble proteins or alcohol-extractable proteins) (Weiss et al. 1992, 1993) should be analyzed.
Lysis of different types of cells and tissues may be achieved by homogenizers (e.g. Potter-homogenizer), liquid nitrogen-cooled mortar and pestle technique, ultrasonic desintegrators, enzymatic lysis (e.g. with lysozyme), detergents (e.g. NP-40, Triton X-100, CHAPS, SDS), osmotic shock, repeated freezing and thawing, or a combination of these methods.
After cell lysis, it is necessary in most cases to inactivate interfering substances (e.g. plant phenols or nucleic acids) and to remove insoluble components by high-speed centrifugation. Plant phenols tend to adsorb proteins and cause distorted protein patterns, horizontal and vertical streaks. Remedies are precipitation of proteins with TCA (20%) and removal of phenols by rinsing with ice-cold aceton/TCA, or to adsorb phenols to (insoluble) polyvinylpolypyrrolidone. If the cells contain high amounts of nucleic acids, the samples may be treated with a protease-free DNAse/RNAse mixture.
Proteases present within samples can produce artifactual spots on 2D maps. It is often recommended to add protease inhibitors (e.g. PMSF, Pefabloc, EDTA, pepstatin, or protease inhibitor coctails), but it must be kept in mind that such reagents can also modify proteins and introduce charge artifacts (Dunn 1993). Other possibilities for protease inactivation are protein precipitation with ice-cold trichloroacetic acid (TCA) or boiling with SDS sample buffer, but it should always be remembered that several proteases can only be inactivated with great difficulty.
After cell lysis and inactivation of interfering substances, proteins have to be solubi-lized. The most widely used solubilization procedure is that based on O‘Farrell (1975), using a mixture of 2% NP-40, 9 M urea, 1% DTT and 0.8% carrier ampholytes ("Lysis buffer"). Instead of NP-40, the non-ionic detergent Triton X-100 or the zwitterionic deter-gent CHAPS are often preferred. Although these methods give ecxellent results in many cases, not all protein complexes are fully disrupted by this mixture. In contrast, the anionic detergent SDS disrupts most non-covalent protein interactions, but cannot be applied in IEF gels. However, SDS can be used as a pre-solubilization procedure for samples prior to IEF, where the sample is initially solubilized in 1% SDS and then diluted with a five-fold volume of Lysis buffer to displace the SDS from the proteins and replace it with a non-ionic or zwitterionic detergent, thereby maintaining the proteins in a soluble state (Dunn 1993).
For the solubilization of hydrophobic proteins, mixtures of urea and thiourea and other detergents than NP-40 (such as sulfobetains) have been recommended (Rabilloud et al. 1997).
Protein extracts should not be too diluted to avoid loss of protein due to adsorption to the wall of the vessel (glass or plastic). The minimum protein concentration should not be less than 0.1 mg/ml, and optimum concentration is 1-5 mg/ml. If samples are rather diluted and contain relatively high concentrations of salts which can interfere with IEF, samples may be desalted. Alternatively, proteins can be precipitated with ice-cold TCA / acetone to remove salts. Diluted samples with a low salt concentration may also be applied directly without further treatment, if the dried IPG strips are reswollen in sample solution. In this case, solid urea, CHAPS and DTT are added to the sample until the desired concentation is obtained (Rabilloud et al. 1994)
Short-time storage (several hours to overnight) of extracts is often possible in the refrigerator (4°C). For a longer time, storage in a freezer or on dry-ice at -78°C is preferred. However, repeated freezing and thawing of the sample must be avoided. Better make portions (aliquots) and thaw only once!
CHAPS, dithiothreitol (DTT) (Sigma), Serdolit MB-1 (Serva), urea, Pefabloc
R (Merck), Pharmalyte (pH 3-10) (Amersham Pharmacia Biotech).
Lysis buffer
(9.5 M urea, 2% (w/v) CHAPS, 0.8% (w/v) Pharmalyte pH 3-10, 1% (w/v) dithiothreitol (DTT) and 5 mM Pefabloc)To prepare 50 ml of Lysis buffer, dissolve 30.0 g of urea in deionized water and make up to 50 ml. Add 0.5 g of Serdolit MB-1, stir for 10 min and filter. Add 1.0 g CHAPS, 0.5 g DTT, 1.0 ml of Pharmalyte pH 3-10 (40% w/v) and, immediately before use, 50 mg Pefabloc proteinase inhibitor to 48 ml of the urea solution. Lysis buffer should always be prepared freshly. Alternatively, make small aliquots (1 ml) and store at -78° C for up to several months..
Notes: a) Lysis buffer thawed once should not be refrozen again! b) Never heat urea solutions above 37 °C in order to reduce the risk of protein carbamylation!
In general, an adequate amount of sample (e.g. yeast or mammalian cells or ground plant seeds) is suspended in Lysis buffer, disrupted by sonification in an ice bath (3x10 sec.) and centrifuged (60 min, 42000 g, 15°C): Yeast cells (120 mg), myeloblasts (5x10
8 cells) or human or animal tissue such as liver or heart (50-100 mg), or ground seeds from legumes (20 mg) or cereals (50-100 mg) are homogenized in a liquid nitrogen cooled mortar. The powder is then suspended in Lysis buffer (1 ml) so that the final protein concentration lies between 5 and 10 mg/ml. Plant tissue proteins (e.g. leaf proteins) are treated with cold TCA/acetone (-20°C) to remove phenolic compounds: Leaves are crushed in a liquid nitrogen cooled mortar, and the powder is resuspended in a pre-cooled (-20°C) solution of 10% trichloroacetic acid (TCA) in acetone with 0.07% 2-mercaptoethanol. Proteins are allowed to precipitate over night at -20°C. After centrifugation the pellet is washed with ice-cold acetone containing 0.07% 2-mercapto-ethanol. The supernatant is discarded and the pellet dried in vacuo.It is also possible to specifically extract certain protein fractions only, e.g. water-soluble (albumins) or alcohol-soluble proteins (gliadins) from cereals and to dilute these extracts with Lysis buffer prior to IPG-IEF (Weiss et al. 1992, 1993).
Protein extracts are either used immediately or are stored at - 78 °C. For analytical runs, typically twenty µl of sample solution are applied onto a single IPG gel strip, whereas for micropreparative runs up to several hundred microliters can be applied, portion by portion. The amount of protein to be loaded onto a single IPG gel strip (separation distance: 180 mm) varies between 50-100 µg for analytical, and 0.5-10 mg for micro-preparative runs, respectively. Alternatively, the sample can be applied directly by in-gel rehydration.