Protein gels isoelectric point

Use of Complex Standards for Cell Proteins and Two-Dimensional Electrophoresis. One of the more widely-used cell lines chosen as a reference standard is the lymphoblastoid cell line GM607, derived from a normal individual and available from the Human Genetic Mutant Cell Depository, Camden, NJ 08103. This cell line may be grown in defined media, labeled with a radioactive tracer, and reproducibly separated in a 2-DE system. Heat shock proteins may readily be isolated and visualized from this cell line, as shown by Anderson et al. (42). For serum, a reference preparation for serum proteins is available as a certified reference material prepared and assayed by the College of American Pathologists (CAP) and by the U. S. Centers for Disease Control. A widely available human serum standard is that provided by the National Bureau of Standards as SRM 909. If sufficient interest from the user community is evident, a full electrophoretic characterization of this material can be included in the documentation. If the amount of selected standard proteins loaded on a gel is known, "relative" quantification of similar proteins could be obtained. In addition, the National Bureau of Standards could serve as an impartial evaluator of potential national standards (e.g. molecular weight standards, "tie-point" proteins, and Isoelectric point standards) to assess suitability and stability. 

Analyses in single and two-dimensional gels (isoelectric point versus molecular weight) have proved to be essential for the analysis of cellular proteins and detailed protocols for the analysis of novel cellular antigens using monoclonal antibodies are given in Chapter 12. 

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

Two-dimensional gel electrophoresis (2DE) is a two-dimensional technique for protein separation, which combines isoelectric focusing and sodium dodecyl sulphate (SDS) electrophoresis. The high resolving power results from separation according to charge (isoelectric point) in the first dimension and size (mobility in a porous gel) in the second dimension. Depending on the gel size, from several hundred to more than 5,000 proteins can be separated. 

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

Innovations in separation science continued on this theme and provided one of the most powerful separation techniques used in biochemistry, where proteins are separated with isoelectric focusing (IEF) applied in one direction, and gel electrophoresis (GE) applied at aright angle to the first separation direction (O Farrell, 1975 Celis and Bravo, 1984). In this case, proteins are first separated according to their isoelectric point, measured in p/units, and then according to their molecular weight by gel electrophoresis. The size separation step is usually aided by addition of a surfactant, most typically sodium dodecyl sulfate (SDS), and the gel material is a polyacrylamide formulation. 

The immobilization procedure performed in two stages allows one to exclude the detrimental effect of acid on the entrapped proteins [44,71,86]. It was demonstrated [87] by the example of a set of oxidases that their activity was retained if the entrapment was carried out at a pH as close to their isoelectric point (pi) as possible. Because the pi value of most enzymes is in the neutral region, the two-stage procedure favors the retention of their functionality. Therefore, the pH shift to the optimal region provides a means of extending the sol-gel entrapment to a wide range of enzymes [45,71]. 

RNase A is a basic protein with an isoelectric point (pi) of 9.45 and a net positive charge in neutral solution.35 However, the conversion of positively charged lysine side chains to polar, but neutrally charged, methylol adducts would be expected to lower the pi of formalin-treated RNase A. To explore this further, RNase A was treated with 5% formalin and analyzed by isoelectric focusing (IEF) gel electrophoresis. Figure 15.5a shows that the pi values were shifted into the pH 6.0-7.4 range. Figure 15.5b shows the results of IEF... 

In your notebook, make a drawing of the observed banding pattern, labeling both the hemoglobin and cytchrome C. Explain, in relevant detail, what you can determine about the isoelectric points of these proteins and what might happen in the gel if the buffer pH were changed from 7.9 to 3.9. 

Proteins can be separated using the 2D electrophoresis method. The first dimension is separation according to the pH of the proteins. The proteins are placed on a gel strip in a buffer solution. An electrical current is applied and the proteins separate and migrate to their isoelectric points (pl)-... 

As with SDS-PAGE gel methods, gel-based isoelectric focusing (lEF) methods have been used for decades to determine isoelectric point pi), which is an intrinsic property of protein molecules. Some complex proteins have multiple charge isoforms with multiple isoelectric points. These isoforms are separated as multiple bands in the lEF gel method. However, like other gel method, the lEF gel has limitations it is not automated, not reproducible, and not quantitative for pi determination. It is also labor intensive and requires large volumes of toxic reagents for staining. 

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