Fast protein analysis

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

The most prominent field of applications for microchip—MS concerns identification and analysis of large molecules in the field of proteomics according to the reduced separation time compared to conventional approaches such as gel-based methods for protein analysis. High-throughput analyses, with lower contamination and disposability, are other features of microfabricated devices that allow the fast screening of proteomic samples in the clinical field. Applications also include the analysis of low-molecular-weight compounds such as peptides or pharmaceutical samples. 

Neumann, H. and Mullner, S. (1998) Two replica blotting methods for fast immunological analysis of common proteins in two-dimensional electrophoresis. Electrophoresis 19, 752-757. 

The introduction of "fast HPLC" has proven to be particularly valuable in protein analysis. As stated earlier, assay time in RP-HPLC analysis of proteins is typically long compared to that for smaller organic molecules. We have evaluated the use of 0.6-cm ID x 4-cm columns packed with 3-um particles in the analysis of insulin by RP-HPLC for potency determination, related substances, and in peptide mapping (7). The use of the "fast column" allows considerable savings (40-60%) in analysis time, compared to the regular (0.46-cm ID x 25-cm) columns, without loss in resolving power. 

Jost, R. 1993. Functional characteristics of dairy proteins. Trends Food Set Technol. 4, 283-288. Juilleart, M.A., Berrocal, R., Chanton, S., Scherz, J.-C., and Jost, R. 1989. Tryptic phosphopeptides from whole casein. 1. Preparation and analysis by fast protein liquid chromatography. J. Dairy Res. 56, 603-611. 

A variant of HPLC is FPLC (fast protein liquid chromatography) (Sheehan 1996), a form of chromatography in which the apparatus and column material is designed specifically with protein analysis and purification in mind. However, this does not imply that conventional HPLC with the right columns is unsuitable for protein chromatography. 

In our laboratory, we rely heavily on 96-well protein precipitation as the primary form of sample preparation. Not only have we found this approach to be the mostuniversal, butitis also the most cost-effective method for discovery bioanalysis. To overcome limitations associated with protein precipitation, CS methods were developed (Figure 11.3) to permit on-line sample cleanup [49]. In addition, fast gradient elution has been used with this strategy to allow expedient method development and fast sample analysis. 

The primary process of lEC involves adsorption and desorption of ionic species from ionogenic groups located in the packing. lEC was the first of the traditional column techniques to be exploited for modern LC, due principally to the need for the fast routine analysis of amino acids and protein mixtures. 

The following sections will describe some of the various methods of liquid chromatography suitable for separation and analysis of biological (macro)molecules. Such systems often use high pressures and rapid flow rates, and are sometimes loosely described as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography). 

When a large electric field is applied across a cell, the transmembrane potential is disrupted and pores are formed on the surface of the membrane. This phenomenon is called electroporation and is often used for gene transfection. As conventionally implemented, the process is reversible, and when the electric field is terminated, the pores close. The phenomenon can also be used to cause permanent disruption of the membrane, effectively lysing the cell. There have been several reports (Ml the use of electric lysis techniques in micM ofluidic devices [9-11]. Of particular interest, fast lysis of individual cells ( 33 ms) by electric pulses for chemical cytometry was demonstrated in a micM ofluidic platform [12]. These extremely rapid lysis methods which minimize unwanted effects of slow lysis (that may bias the results) make these techniques favorable for protein analysis when compared to chemical lysis techniques. One drawback of electric lysis is that much of the cell membrane, subcellular structures, and the nucleus may remain intact and thus can clog the channel or adhere to the surface, affecting the separation and limiting the capacity for reuse. 

In recent years, many analytical strategies for elemental speciation analysis, particularly in (bio)medi-cal speciation, make use of LC-atomic detector hybrid techniques. Most frequently studied elements include As, Pb, Cd, Sn, Hg, Se, Cr, and A1 both in biological and environmental samples. The capability for multielemental detection of ICP-MS can be most useful in such analysis. An illustrative example of multielement trace-element speciation is metal-lothionein proteins (MT) from rabbit liver by LC-ICP-MS. Comparative profiles for Cd, Zn, Cu, and S obtained for MTs from rabbit liver, using a fast protein liquid chromatography (FPLC) column coupled to ICP-MS are shown in Figure 7, using molecular and specific detectors. 

Andrews, A.T. Taylor, M.D. Owen, A.J. Rapid analysis of bovine milk proteins by fast protein liquid chromatography. J. Chromatogr. 1985, 348,177-185. 

Figure 2. The fast protein liquid chromatographic (FPLC) analysis of isoenzymes isolated from the liver of rats treated with cottonseed oil (Control), 2-n-butylthiophene (BT), and 2-n-heptylfuran (HF).

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