TECHNIQUES FOR SAMPLE PREPARATION Dialysis

One of the oldest procedures applied to the purification and characterization of biomolecules is dialysis, an operation used to separate dissolved molecules on the basis of their molecular size. The technique involves sealing an aqueous solution containing both macromolecules and small molecules in a porous membrane.

The sealed membrane is placed in a large container of low-ionic-strength buffer.

The membrane pores are too small to allow diffusion of macromolecules of molec-ular weight greater than about 10,000. Smaller molecules diffuse freely through the openings (Figure 3.7). The passage of smaller molecules continues until their concentrations inside the dialysis tubing and outside in the large volume of buffer are equal. Thus, the concentration of small molecules inside the membrane is reduced. Equilibrium is volume dependent and is reached after 4 to 6 hours.

Of course, if the outside solution (dialysate) is replaced with fresh buffer after equi-librium is reached, the concentration of small molecules inside the membrane will be further reduced by continued dialysis.

Dialysis membranes are available in a variety of materials and sizes. The most common materials are collodion, cellophane, and cellulose. Recent

Membrane

Inside Outside

FIGURE 3.7 Diffusion of smaller molecules through dialysis membrane pores.

modifications in membrane construction make a range of pore sizes available.

Spectrum Laboratories offers Spectra/Por membrane tubing with complete molecular weight cutoffs ranging from 100 to 300,000.

Dialysis is most commonly used to remove salts and other small molecules from solutions of macromolecules. During the separation and purification of biomolecules, small molecules are added to selectively precipitate or dissolve the desired molecule. For example, proteins are often precipitated by addition of organic solvents or salts such as ammonium or sodium sulfate. Since the presence of organics or salts usually interferes with further purification and characterization of the molecule, they must be removed. Dialysis is a simple, inexpensive, and effec-tive method for removing all small molecules, ionic or nonionic.

Dialysis is also useful for removing small ions and molecules that are weakly bound to biomolecules. Protein cofactors such as NAD, FAD, and metal ions can often be dissociated by dialysis. The removal of metal ions is facilitated by the addition of a chelating agent (EDTA) to the dialysate.

Dialysis of small-volume samples (less than 3 mL) is often tedious, incon-venient, and inefficient. Commercial vials for dialysis are now available for sam-ple sizes from 0.05 to 3.0 mL, with molecular weight cutoffs ranging from 3500 to 14,000 daltons. For example, GeBAflex tubes, offered by Gene Bio-Applications, may be used for dialysis (the removal of small molecules like urea, detergents, and ethidium bromide), buffer exchange, sample concentration, and electroelu-tion of protein and nucleic acid samples (see Chapter 6).

Ultrafiltration

Although dialysis is still used occasionally as a purification tool, it has been largely replaced by ultrafiltration and gel filtration (see Chapter 5, Section E, p. 132). The major disadvantage of dialysis that is overcome by the newer meth-ods is that it may take several days of dialysis to attain a suitable separation. The other methods require 1 to 2 hours or less.

Ultrafiltration involves the separation of molecular species on the basis of size, shape, and/or charge. The solution to be separated is forced through a membrane by an external force. Membranes may be chosen for optimum flow rate, molecular specificity, and molecular weight cutoff. Two applications of membrane filtration are obvious: (1) desalting buffers or other solutions, and (2) clarification of turbid solutions by removal of micron- or submicron-sized particles. Other applications are discussed below.

Ultrafiltration membranes have molecular weight cutoffs in the range of 100 to 1,000,000. They are usually composed of two layers: (1) a thin (0.1–0.5 ), surface, semipermeable membrane made from a variety of materials including cellulose acetate, nylon, and polyvinylidene, and (2) a thicker, inert, support base (Figure 3.8). These filters function by retaining particles on the surfaces, not within the base matrix.

Membrane filters of these materials can be manufactured with a predeter-mined and accurately controlled pore size. Filters are available with a mean pore size ranging from 0.025 to These filters require suction, pressure, or cen-trifugal force for liquid flow. A typical flow rate for the commonly used

membrane is 57 mL min^-1cm^-2at 10 psi.

0.45–mm 15 mm.

mm

Ultrafiltration devices are available for macroseparations (up to 50 L) or for microseparations ( to microliters). For solutions larger than a few milli-liters, gas-pressurized cells or suction-filter devices are used. For concentration and purification of samples in the milli- to microliter range, disposable filters are available. These devices, often called microconcentrators, offer the user simplicity, time saving, and high recovery. The sample is placed in a reservoir above the membrane and centrifuged. The time and centrifugal force required depend on the membrane, with spin times varying from 30 minutes to 2 hours and forces from to Figure 3.9 outlines the use of a centrifuge microfilter.

The principles behind ultrafiltration are sometimes misunderstood. The nomenclature implies that separations are the result of physical trapping of the particles and molecules by the filter. With polycarbonate and fiberglass filters, separations are made primarily on the basis of physical size. Other filters (cellu-lose nitrate, polyvinylidene fluoride, and to a lesser extent cellu(cellu-lose acetate) trap particles that cannot pass through the pores, but also retain macromolecules by adsorption. In particular, these materials have protein- and nucleic acid-binding properties. Each type of membrane displays a different affinity for various molecules. For protein, the relative binding affinity is polyvinylidene fluoride cellulose nitrate cellulose acetate. We can expect to see many applications of the

“affinity membranes” in the future as the various membrane surface chemistries are altered and made more specific. Some applications are described in the following pages.

Clarification of Solutions

Because of low solubility and denaturation, solutions of biomolecules or cellular extracts are often turbid. This is a particular disadvantage if spectrophotometric analysis is desired. The transmittance of turbid solutions can be greatly increased by passage through a membrane filter system.

7 7

7500 * g.

1000 * g

FIGURE 3.8 Electron

micrograph of an ultrafiltration membrane showing the two layers.

Particles greater than in diameter are retained on the surface or within pores. Courtesy of the Millipore Corporation; www.

millipore.com.

0.1 mm

This simple technique may also be applied to the sterilization of nonau-toclavable materials such as protein and nucleic acid solutions or heat-labile reagents. Bacterial contamination can be removed from these solutions by passing them through filter systems that have been sterilized by autoclaving.

1. Pipet up to 2 ml of sample into the top reservoir.

2. Cover with the cone-shaped cap and spin in a centrifuge with a fixed-angle rotor.

3. Detach and invert top half.

4. Centrifuge 1 to 2 minutes to spin the concentrate into the cone-shaped cap.

5. Concentrate is now completely accessible for further

analysis. Both concentrate and filtrate can be stored in collection cups.

FIGURE 3.9 Use of a centrifuge microfilter.

Courtesy of the Millipore

Corporation; www.

millipore.com.

Collection of Precipitates for Analysis

The collection of small amounts of very fine precipitates is the basis for many chemical and biochemical analytical procedures. Membrane filtration is an ideal method for sample collection. This is of great advantage in the collection of radioactive precipitates. Cellulose nitrate and fiberglass filters are often used to collect radioactive samples because they can be analyzed by direct suspension in an appropriate scintillation cocktail.

Harvesting of Bacterial Cells from Fermentation Broths

The collection of bacterial cells from nutrient broths is typically done by batch centrifugation. This time-consuming operation can be replaced by membrane filtration. Filtration is faster than centrifugation, and it allows extensive cell washing.

Concentration of Biomolecule Solutions

Protein or nucleic acid solutions obtained from extraction or various purifica-tion steps are often too dilute for further investigapurifica-tion. Since they cannot be con-centrated by high-temperature evaporation of solvent, gentler methods have been developed. One of the most effective is the use of ultrafiltration pressure cells, as shown in Figure 3.10. A membrane filter is placed in the bottom and the solution is poured into the cell. High pressure, exerted by compressed nitrogen (air could cause oxidation and denaturation of biomolecules), forces the flow of small molecules, including solvent, through the filter. Membranes are available in a number of sizes, allowing a large variety of molecular weight cutoffs. Larger molecules that cannot pass through the pores are concentrated in the sample chamber. This method of concentration is rapid and gentle and can be performed at cold temperatures to ensure minimal inactivation of the molecules. One major disadvantage is clogging of the pores, which reduces the flow rate through the filter; this is lessened by constant but gentle stirring of the solution.

Lyophilization and Centrifugal Vacuum Concentration

Although ultrafiltration is being used more and more for the concentration of biological solutions, the older technique of lyophilization (freeze-drying) is still used. There are some situations (storing or transporting biological materials) in which lyophilization is preferred. Lyophilization is a drying technique that uses the process of sublimation to change a solvent (water) in the frozen state directly to the vapor state under vacuum. The product after lyophilization is a fluffy matrix that may be reconstituted by the addition of liquid. This is one of the most effective methods for drying or concentrating heat-sensitive materials. In practice, a biological solution to be concentrated is “shell-frozen” on the walls of a round-bottom or freeze-drying flask. Freezing of the solution is accomplished by placing the flask (half full with sample) in a dry ice–acetone bath and slowly rotating it as it is held at a angle. The aqueous solution freezes in layers on the wall of the flask. This provides a large surface area for evaporation of water. The flask is then connected to the lyophilizer, which consists of a refrigeration unit and a vacuum pump (see Figure 3.11). The combined unit maintains the sample

45°

at for stability of the biological materials and applies a vacuum of approx-imately 5 to 25 mm Hg on the sample. Ice formed from the aqueous solution sub-limes and is pumped from the sample vial. In fact, all materials that are volatile under these conditions ( 5 to 25 mm Hg) will be removed, and nonvolatile materials (proteins, buffer salts, nucleic acids, etc.) will be concentrated into a light, sometimes fluffy precipitate. Most freeze-dried biological materials are stable for long periods of time and some remain viable for many years.

As with any laboratory method, there are precautions for and limita-tions of lyophilization that must be understood. Only aqueous solulimita-tions should be lyophilized. Organic solvents lower the melting point of aqueous solutions and increase the chances that the sample will melt and become denatured dur-ing freeze-drydur-ing. There is also the possibility that organic vapors will pass through the cold trap into the vacuum pump, where they may cause damage.

A new and increasingly popular technique for sample concentration and drying is centrifugal vacuum concentration. The method may be used to dry a wide variety of biological samples. The process starts with a sample dissolved in a solvent (water or organic). The solvent is evaporated under vacuum in a centrifuge, thus producing a pellet in the bottom of the container. The most widely used

-40°C, -40°C

Pressure inlet

Pressure relief valve

Transparent body

Stirring bar

Beaker design

O-ring Membrane

Membrane support Filtrate outlet FIGURE 3.10

Schematic of an ultrafiltration cell.

Courtesy of Millipore/Amicon Corporation; www.

millipore.com.

instrument is the SpeedVac, available through Savant (Figure 3.12). This method is better than freeze-drying because it is faster, it does not require a prefreezing step, it provides 100% sample recovery, and it may be used with solvents other than water.

The SpeedVac is usually used for relatively small volumes—1–2-mL samples.

FIGURE 3.11 A lab technician prepares a sample for lyophilization (freeze-drying) with the Labconco FreeZone benchtop freeze dryer, Model

# 7740020. © Labconco

Corporation. Photo used with

permission from Labconco

Corporation. www.

labconco.com.

FIGURE 3.12 The SpeedVac

centrifugal vacuum concentrator.

Image provided by Thermo Fisher Scientific Inc., all rights reserved.

www.thermofisher.

com.