Sample Application

The protein application mode can affect the amount of protein entering the IPG strip during IEF. There are several ways of applying the sample. In the system supplied by Amersham Pharmacia Biotechnology (Multiphor II), the sample is usually loaded into application cups (also supplied by Amersham Pharmacia Biotechnology). Up to 150 |L can be applied in one cup. The cups are fixed in special 'cup accommodating bridges' which are placed near the basic or acidic end of the strip. It seems that sample application at the basic end of the strip is more advantageous compared to the application at the acidic end. We have, however, found that simultaneous sample application at both the basic and the acidic ends of the strip can result in the detection of more and stronger protein spots compared to sample application at only one end. It also allows the simultaneous application of sample volumes larger than 150 |L. From a technical point of view, sample application using the cups is the most difficult operation to perform. The cups should touch the polyacrylamide gel on the strip, otherwise the sample will leak; they should also not damage the gel at the contact point, otherwise the proteins will not enter the strip.

An alternative method of sample application is the rehydration of the strip in a solution containing the protein sample. This approach is convenient to perform and theoretically it should result in the detection of all proteins present in the sample. However, more comparative studies are required to prove that this approach is more efficient than the loading of sample into cups. Amersham Pharmacia Biotechnology has recently introduced a new IEF apparatus (IPGphor) in which sample application and IEF can be performed. The strips are placed in special ceramic strip holders and rehydrated for the desired time in a solution containing the protein sample. Each strip holder holds a single IPG strip throughout rehydration and IEF. IEF starts automatically after rehydration according to the conditions programmed. Whether the performance of IEF will be improved with the use of this instrument is not clear at present.

The quantity of protein to be applied on the strip naturally depends on the goal of the analysis. If the identification of protein spots is intended, the amount loaded should be in the order of 1 mg or higher, depending on the number of proteins in the mixture. A 1D gel analysis of the sample prior to 2D elec-trophoresis may provide helpful information as to defining the right protein quantity. If large amounts of protein are applied, a percentage of the proteins may not enter the strip. Presently, this constitutes a drawback of the IPG strip approach. Because certain proteins in the sample (mainly major components) only partially enter the strip, this can result in an unreliable quantification of a particular protein in a given mixture. While the application of 15 mg or more of protein sample has been reported, we consider that 2-4 mg is the limit for a productive separation, using the strips that are presently available.

IEF using IPG strips can separate basic proteins efficiently with pIs up to about 12. The introduction of low concentrations of isopropanol in the rehydra-tion buffer improves focusing of basic proteins. Hy-drophobic proteins probably precipitate at the point of application and efficient separation has not yet been reported. Hydrophobic proteins can be analysed in a different 2D electrophoresis system, which uses the interaction of the proteins with a cationic detergent in the first dimension rather than pi. The second dimension is, as described below, dependent on the molecular mass. The separated proteins form approximately a diagonal line. Relatively, only a small number of proteins can be successfully separated using this approach.

Second-Dimensional Separation (Sodium Dodecyl Sulfate-Polyacryl-amide Gel Electrophoresis, SDS-PAGE)

Following IEF, the proteins are separated according to their molecular masses. During this nonequilib-

rium step, the proteins are negatively charged by addition of the anionic detergent SDS. Upon application of an electric field, the charged proteins move along a porous polyacrylamide gel and are separated according to their size. A reducing agent is also included to disrupt disulfide bonds. In comparison with IEF, SDS-PAGE is relatively easy to control. The terms ISO-DALT and IPG-DALT are often used to mean 2D gel electrophoresis employing IEF with carrier ampholytes or IPG strips, respectively.

Horizontal or more usually vertical slab gels, running in a discontinuous buffer system are employed. A high throughput analysis is facilitated by the use of tanks accommodating 6-20 gels running in parallel. An efficient separation of thousands of proteins present in complex mixtures, can only be performed on gels of a large format (18 x 20 or 25 x 25 cm). Either gradient gels or gels of a constant acrylamide concentration can be used. Because of the complexity of the

Figure 4 Partial 2D gel images showing an improved spot resolution by using different acrylamide concentrations. Separation of rat brain proteins on a 9-16% SDS gel (A) and on a 7.5-16% SDS gel (B). Separation of low molecular mass soluble proteins from H. influenzae on 9-16% SDS gel (C) and on a 10% SDS Tricine gel (D). (B, D) The gel parts comprising the corresponding proteins shown in A and B, respectively, are longer on account of the different acrylamide concentrations.

Figure 4 Partial 2D gel images showing an improved spot resolution by using different acrylamide concentrations. Separation of rat brain proteins on a 9-16% SDS gel (A) and on a 7.5-16% SDS gel (B). Separation of low molecular mass soluble proteins from H. influenzae on 9-16% SDS gel (C) and on a 10% SDS Tricine gel (D). (B, D) The gel parts comprising the corresponding proteins shown in A and B, respectively, are longer on account of the different acrylamide concentrations.

technology and the large diversity of the samples to be analysed, and in order for the data to be useful to a broad research community, 2D PAGE has been to a large extent standardized. In the second dimension, for example, we usually use 9-16% linear gradient gels. This gel system represents a good compromise, as it separates proteins between 5 and 200 kDa. However, efficient separation is limited to a range of approximately 15-40 kDa. Outside this range, in particular above 50 and below 10 kDa, the separation is often suboptimal. For more efficient separation, gels of a different acrylamide concentration should be tried. Figure 4 gives examples of the improved separation of high molecular mass brain proteins using gels of lower acrylamide concentration and of low molecular mass proteins from Haemophilus influenzae, using gels with Tricine as the trailing ion instead of Tris.

For spot visualization, the gels can be stained with either silver or Coomassie blue (usually colloidal Coomassie blue), depending on the quantity of pro tein sample applied and the aim of analysis. Silver stain may be preferentially used for gel comparison studies, whereas staining with Coomassie is preferred when the spots are intended for protein identification. Colloidal Coomassie blue has the advantage that the stain is sensitive enough and the gels can be easily destained with water. The simultaneous staining of many gels in one tank substantially increases the throughput. Apart from silver and Coomassie blue, several other protein detection methods exist, such as staining with various metals, labelling with fluorescent agents or detection of radiolabelled compounds, after gel drying and exposure, for example to a film.

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