Which component of an electrophoresis system sets the pH?
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When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will initially move toward the electrode with the opposite charge. During migration through the pH gradient, the protein will either pick up or lose protons. As it migrates, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point where the pH gradient is equal to its pI. There, being uncharged, it will stop migrating (see figure below). If this protein should happen to diffuse to a region of lower pH, it will become protonated and be forced back toward the cathode by the electric field. If, on the other hand, it diffuses into a region of pH greater than its pI, the protein will become negatively charged and will be driven toward the anode. In this way, proteins condense, or are focused, into sharp bands in the pH gradient at their individual characteristic pI values. Show Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective pI values at differing rates, but remain relatively fixed at those pH values for extended periods. By contrast, proteins in conventional electrophoresis continue to move through the medium until the electric field is removed. Moreover, in IEF, proteins migrate to their steady-state positions from anywhere in the system.  In IEF, a mixture of proteins is resolved on a pH 3–10 IPG strip according to each protein's pI and independently of its size. A stable, linear, and reproducible pH gradient is crucial to successful IEF. IPG strips offer the advantage of gradient stability over extended focusing runs (Bjellqvist et al. 1982). IPG strips are much more difficult to cast than carrier ampholyte gels (Righetti 1983); however, IPG strips are commercially available (see ReadyStrip IPG Strips for available gradients and sizes). pH gradients for IPG strips are created with sets of acrylamido buffers, which are derivatives of acrylamide containing both reactive double bonds and buffering groups. The general structure is CH2=CH–CO–NH–R, where R contains either a carboxyl [–COOH] or a tertiary amino group (e.g., –N(CH3)2). These acrylamide derivatives are covalently incorporated into polyacrylamide gels at the time of casting and can form almost any conceivable pH gradient (Righetti 1990). ReadyStrip IPG Strips Relative
focusing power of IPG strips
Use of broad range strips (pH 3–10) allows the display of most proteins in a single gel. With narrow range and micro range overlapping gradient strips, resolution is increased by expanding a small pH range across the entire width of a gel. Since many proteins are focused in the middle of the pH range 3–10, some researchers use nonlinear (NL) gradients to better resolve proteins in the middle of the pH range and to compress the width of the extreme pH ranges at the ends of the gradients. However, overlapping narrow range and micro range linear IPG strips can outperform a nonlinear gradient and display more spots per sample (see figure below). 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 gels by matching spots from the overlapping regions using imaging software. Proteins that are outside of the pH range of the strip are excluded, therefore, more total protein mass can be loaded per strip, allowing more proteins to be detected. The figure below demonstrates the resolution achieved using 3 overlapping gradients with 17cm ReadyStrip IPG strips and large format gels.
Narrow overlapping IPG strips outperform nonlinear IPG strips. Proteins were separated by 2-D PAGE using 17 cm IPG strips, then stained with Coomassie Blue. A, 2 mg protein on pH 3–6 ReadyStrip IPG; B, 2 mg protein on pH 5–8 ReadyStrip IPG; C, 1 mg protein on pH 7–10 ReadyStrip IPG; D, 0.8 mg protein on a nonlinear pH 3–10 IPG. With the narrow range strips (A–C), more sample was loaded, and consequently, more spots were resolved and detected after staining compared to the wide-range IPG strip (D). Kindly provided by Sjouke Hoving, Hans Voshol, and Jan van Oostrom of Novartis Pharma AG, Functional Genomics Area, Basel, Switzerland. The 17, 18, and 24 cm IPG strips and large format gels have a large area to resolve proteins 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, such as Bio-Rad's Mini-PROTEAN® 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 when choosing gel size. The table below compares gel size, run times, and equipment. The ability to cast or run 12 gels at a time in any 3 size formats is very useful in gathering proteomic results. In some cases, mini systems (7cm ReadyStrip IPG strips with Mini-PROTEAN® precast gels, or 11cm 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 to 44 cm. 2-D Dimension Instrument Selection Chart
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. The easiest and most efficient way of applying the sample is while rehydrating the strip. In some specific instances, it is best to rehydrate the strips and then apply sample through sample cups while current is applied. Sample Application During Rehydration
Whether the strips are hydrated actively or passively, it is very important that they be incubated with sample for at least 12 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 stead-state technique, so proteins migrate to their pI independent of their initial positions. The advantages of this approach are:
Sample Application By Cup Loading Cup loading can be beneficial in the following cases:
During an IEF run, the electrical conductivity of the gel changes with time, especially during the early phase. When an electric field is applied to an IPG at the beginning of an IEF run, the current will be relatively high due to the large number of charge carriers present. As the proteins and ampholytes move toward their pIs, the current will gradually decrease due to the decrease in the charge on individual proteins and carrier ampholytes. The pH gradient, strip length, and the applied electric field determine the resolution of an IEF run. According to both theory and experiments, the difference in pI between two adjacent IEF-resolved protein bands is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient at the position of the bands (Garfin 2000). Thus, narrow pH ranges and high applied voltages yield higher resolution in IEF. The highest resolution can be achieved using micro-range IPG strips and an electrophoretic cell, such as the PROTEAN i12 IEF system, capable of applying high voltages. IEF runs should always be carried out at the highest voltage compatible with the IPG strips and electrophoretic cell. At the completion of focusing, the current drops to nearly zero since the carriers of the current have stopped moving. The PROTEAN i12 IEF system is designed to provide precise cooling, allowing the highest possible voltages to be applied. The PROTEAN i12 IEF system offers individual lane control allowing running different samples, pH gradients and protocols to be run simultaneously. A traditional IEF cell sets a current limit that is averaged among the lanes. If one sample has higher conductivity, it will pull more current which can result in over-focusing or even burning the IPG strip. This can also result in under-focusing of the lower conductivity sample resulting in a streaky 2D gel. It is recommended when running traditional IEF cells that the samples are very similar in conductivity and the pH ranges of the strips be identical to avoid these problems. With the PROTEAN i12 IEF system, each lane is controlled independently and the current limit can be set for each lane. A current limit for each sample can be reliably set and will not vary based on the other samples in the experiment. This will result in more consistent and reproducible results. A default current limit of 50 µA per strip is intended to minimize protein carbamylation reactions in urea sample buffers. This limit can be increased to 100 µA per strip if desired. Given that the pH gradient is fixed in the IPG strip gel, focused proteins are more stable at their pI than in conventional IEF gels. Focused IPG strips can be stored at –20°C indefinitely without affecting the final 2-D pattern. IPG strips are bound to a plastic sheet, so gel cracking, which results from expansion and contraction during freezing and thawing, is avoided and the IPG strips retain their original dimensions after thawing. It is convenient to store IPG strips in rehydration trays, which can then be used to equilibrate the strips for the second dimension. Which electrophoresis method first creates a pH gradient across the gel before migration of proteins?Which electrophoresis method first creates a pH gradient across the gel before migration of proteins? a. In isoelectric focusing, the ampholytes migrate first and create a pH gradient across the gel. Then the proteins migrate to the pH area that matches their isoelectric point.
Why does solution pH affect separation of proteins by electrophoresis?The net charge of a protein depends on the pH of its environment. In a solution with a pH above its isoelectric point, a protein has a net negative charge and will migrate towards the positive electrode.
What is the ideal buffer pH for serum protein electrophoresis?Clinical laboratories most commonly use barbital buffers at pH 8.6 to perform the serum protein electrophoresis (SPEP) test. Under these conditions immunoglobulins have little net charge and tend to be influenced by endosmosis.
What are the parameters of electrophoresis separation depend on?Electrophoretic conditions are characterized by the electrical parameters (current, voltage, power), and factors such as ionic strength, pH value, viscosity, pore size, etc., which describe the medium in which the particles are moving.
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