**2.2.8 Sample application during rehydration**

For both active and passive rehydration methods, the sample is introduced to the IPG strip at the time of rehydration. As the strips hydrate, proteins in the sample are absorbed and distributed over the entire length of the strip (Sanchez *et al.* 1997). In the case of active rehydration, a very low voltage is applied during rehydration of the strips. Proteins enter

per sample. This result is due to the extra resolving power from use of a narrower pI range per gel. Use of overlapping gradients also allows the ability to create "cyber" or composite

The 17 cm IPG strips and large-format gels have a large area to resolve protein spots; however, they take a long time to run. Using a mini system instead of, or as a complement to, a large gel format can provide significant time savings. A mini system is perfect for rapid optimization of sample preparation methods. Switching to a large format then allows thorough assessment of a complex sample and identification of proteins of interest. In many cases, a mini system consisting of narrow-range IPG strips can then be used to focus in on the proteins of interest. Throughput of the 2-D process is a consideration in choosing gel size. The ability to cast or run 12 gels at a time in any of 3 size formats is very useful in gathering proteomic results. In some cases, mini systems (7 cm ReadyStrip IPG strips with Mini-PROTEAN® 3 format gels, or 11 cm ReadyStrip IPG strips with Criterion™ precast gels) can completely replace large 2-D systems, providing speed, convenience, and ease in handling. The availability of narrow and micro overlapping pH-range ReadyStrip IPG strips can increase the effective width of pI resolution more than 5-fold after accounting for overlapping regions. When 3 narrow-range overlapping ReadyStrip IPG strips are used with the Criterion system, the resolution in the first dimension is increased from 11 to 26 cm. When micro-range strips are used, the resolution in the first dimension is expanded from 11

The pI of a protein can be estimated by comparing the position of the protein spot of interest to the position of known proteins or standards separated across the same pH gradient (Bjellqvist *et al.* 1993b, Garfin 2000). ReadyStrip IPG strips contain linear gradients, so the pI of an unknown protein can be estimated by linear interpolation relative to proteins of

Commercial IPG strips are dehydrated and must be rehydrated to their original gel thickness (0.5 mm) before use. This allows flexibility in applying sample to the strips. There are 3 methods for sample loading: passive in-gel rehydration with sample, active in-gel rehydration with sample, or cup loading of sample after IPG rehydration. Introducing the sample while the strips are rehydrating is the easiest method. In some specific instances, it is best to rehydrate

For both active and passive rehydration methods, the sample is introduced to the IPG strip at the time of rehydration. As the strips hydrate, proteins in the sample are absorbed and distributed over the entire length of the strip (Sanchez *et al.* 1997). In the case of active rehydration, a very low voltage is applied during rehydration of the strips. Proteins enter

the strips and then apply sample through sample cups while current is applied.

gels by matching spots from the overlapping regions using imaging software.

**2.2.5 IPG strip (2-D array) size** 

to 44 cm.

known pI.

**2.2.6 Estimation of pI** 

**2.2.7 Sample application** 

**2.2.8 Sample application during rehydration** 

the gel matrix under current as well as by absorption. The PROTEAN IEF cell has preprogrammed methods designed to accommodate active rehydration. Active rehydration is thought to help large proteins enter the strip by applying electrical "pull". Because the voltage is applied before all the solution and proteins are absorbed into the gel, the pH of a protein's environment will be the pH of the rehydration buffer, and the protein will move according to its mass-to charge ratio in that environment. Thus, small proteins with a higher mobility have a higher risk of being lost from the strip. With passive rehydration, proteins enter the gel by absorption only. This method allows efficient use of equipment since strips can be rehydrated in sample rehydration trays while other samples are being focused in the IEF cell.

Whether the strips are hydrated actively or passively, it is very important that they be incubated with sample for at least 11 hr prior to focusing. This allows the high molecular weight proteins time to enter the gel after the gel has become fully hydrated and the pores have attained full size. These sample application methods work because IEF is a steady-state technique, so proteins migrate to their pI independent of their initial positions.

The advantages of this approach are:

