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Sample preparation

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 makeup 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.

For the solubilization of more hydrophobic proteins it is recommended to use 7 M urea, 2 M thiourea instead of 9.5 M urea

Lysis buffer thawed once shouldn´t be refrozen again.

Never heat urea solutions above 37 C.
(Otherwise, protein carbamylation may occur.)

Cell Lysis

In general, an adequate amount of deep-frozen cells or tissue (e.g. mouse liver, yeast, plant seeds etc.) is disrupted by different techniques such as grinding under liquid nitrogen with mortar and pestle, sonication with an ultrasonic probe in an ice bath (5 x 5 sec. with intermittant cooling), shearing-based methods, or homogenization. After cell lysis, proteins are solubilized with sonication either (a) in lysis buffer (9 M urea, 1% w/v dithiothreitol, 2-4% w/v CHAPS, 2% v/v carrier ampholytes, pH 3-10 and 10 mM PefablocR proteinase inhibitor), (b) in thiourea lysis buffer (2M thiourea, 7M urea, 2-4% w/v CHAPS, 1% w/v dithiothreitol and 2% v/v carrier ampholytes pH 3-10 and 10 mM PefablocR proteinase inhibitor), or (c) in hot SDS sample buffer (1% w/v SDS, 100 mM Tris-HCl pH 7.0) and then diluted with at least a three-fold excess of lysis buffer or thiourea lysis buffer. All extracts are thoroughly centrifuged (60 min, 40000 g, 15C).

Tissue samples are collected as soon as possible after the death of the "donor" and immediately frozen at 196 C. All samples are disrupted while still frozen. Small tissue specimen are wrapped in aluminium foil, frozen in liquid nitrogen and crushed with a precooled hammer (-20 C), whereas larger tissue pieces are ground under liquid nitrogen using a pestle and mortar, and then solubilized in lysis buffer. E.g., myeloblasts (5x108 cells) or human or animal tissue such as liver or heart (50-100 mg) are homogenized under cooling with liquid nitrogen. The powder is then suspended in Lysis buffer (1 ml) so that the final protein concentration is between 5 and 10 mg/ml. For enrichment of alkaline proteins.

Mouse liver is ground under cooling with liquid nitrogen in a mortar with pestle, suspended in 20% TCA in acetone (-18C) containing 0.2% DTT, and kept at -18C overnight in order to ensure complete protein precipitation. Following centrifugation (40,000 g, 60 min, 15C), the supernatant is discarded and the pellet resuspended in acetone containing 0.2% DTT. The sample is spun again, and the pellet dried under vacuum and then solubilized in lysis buffer (Grg et al. 1999). Following centrifugation, the supernatants are stored in aliquots at -70C until analyzed.

Preparation of total ribosomal proteins is recommended according to Madjar (1994), preparation of histones from chicken erythrocytes according to Csordas et al. (1991)

Microbial cell cultures such as bacteria or yeast need to be standardized and optimized for the growth conditions and the growth phase determined from which the sample is taken, since this has an enormous impact upon the biochemical state of the cells. Because the cells may excrete proteases and other extracellular enzymes into the growth media, they first need to be washed with an isotonic buffer such as phosphatebuffered saline (PBS) or sucrose (at the same temperature as the culture, so as not to cold- or heat-shock the cells) prior to harvesting by centrifugation. Since bacteria and yeast cells are surrounded by a cell wall, protein extraction can not be simply carried out by an osmotic shock as can be done e.g. with mammalian cells. Extensive disruption of cells is required, either by vigorously shaking the cells in the presence of glass beads, or by sonication on ice in presence of (urea/thiourea) lysis buffer, or by heating the sample in the presence of SDS. Where necessary, protease-free DNAse and RNAse is added to digest nucleic acids.

Dry plant seeds (where proteases are not active) are simply smashed with a hammer, and ground with a mortar and pestle (with or without cooling by liquid nitrogen). The plant tissue is then solubilized in lysis buffer, centrifuged and aliquoted. 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).

Plant leaves not only contain proteases, but also high concentrations of phenols which can adsorb proteins and cause streaks on the 2-D electrophoresis gel. To counter this, cells are first disrupted with mortar and pestle in presence of liquid nitrogen. Proteins are then precipitated with 20% TCA in ice-cold acetone identical to the procedure described for mouse liver proteins. After removal of plant phenols by rinsing with ice-cold acetone, proteins are solubilized in lysis buffer, and aliquoted.

Protein extracts are either used immediately or are stored at - 78 C. For analytical runs,typically twenty l 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 g for analytical, and 0.5-10 mg for micropreparative runs, respectively. Alternatively, the sample can be applied directly by in-gel rehydration (Rabilloud et al. 1994, Sanchez et al. 1997).