Protein chips" method

Duan, L., Wang, Y., Li, S.S-C., Wan, Z., and Zhai, J. (2005) Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method. BMC Infect. Dis. 5, 53. http //www.biomedcentral. com/1471-2334/5/53. 

The basic principle behind the ChIP method is relatively simple It is based on the selective enrichment of a chromatin fraction containing a specific antigen (e.g. transcription factors, DNA binding proteins, modified histones, etc.) by an immunoprecipitation step. Specific (important, see below ) antibodies that recognize a protein of interest or the modified form of a protein can be used to determine the relative abundance of it within DNA regions. 

In a more modified approach, differential display proteomics can also be done with no separation of proteins. This is called the protein chip approach. In this method, a variety of bait proteins such as antibodies, peptides, or protein fragments may be immobilized in an array format on specially treated surfaces. The surface is then probed with the samples of interest. Proteins that bind to the relevant target can then be analyzed by direct MALDI readout of the bound material (Nelson, 1997 Davies et ah, 1999). Lor example, well-characterized antibodies can be used as bait. Protein samples from two different cell states are then labeled by different fluorophores, mixed together, and used as probe. In such a case, the fluorescent color acts as an indicator for any change in the abundance of the protein that remains bound to the chip (Lueking et ah, 1999). A number of technical problems would still need to be overcome before applying this technique for large-scale analysis of proteins. 

First, a general method of transformation of the expression plasmid into E. coli will be described, as well as appropriate conditions for growth and induction of the expression cultures. Then, the 96-well protein purification method is detailed. Last, analysis of the purified proteins is described using both lab-on-a-chip technology and traditional sodium dodecyl sulfide (SDS)-polyacryla-mide gel electrophoresis (PAGE). 

Warren EN, Elms PJ, Parker CE, et al. Development of a protein chip a MS-based method for quantitation of protein expression and modification levels using an immunoaffmity approach. Anal. Chem. (2004) 76 4082-4092. 

So far the bottleneck in producing protein chips seems to be the preparation of the individual proteins, but for this heavily researched area solutions are on the horizon. The advantage of the protein chip approach is that a comprehensive set of individual proteins can be directly screened in vitro for a wide variety of activity, including protein-drug interactions, protein-lipid interactions, and enzymatic assays using a wide range of in vitro conditions - and faster and cheaper than with conventional methods. 

SELDI-TOF is beginning to offer an alternative to 2DGE. Surface-enhanced laser desorption/ionizitation (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively adsorbed to a chemically modified surface on a biochip (Ciphergen Protein Chip Arrays). 

Another method using protein "chips has also been developed (Haab et al., 2001 Zhao et al., 2001 Zhu et al., 2001). These chips contain ordered arrays of protein-binding molecules with the proteins then bound or enriched on a segment of the chip to be analyzed via various technologies. Antibodies recognizing specific protein sequences or modifications can be used, for example, to separate proteins with a specific activity from a complex mixture. A different version of a protein chip has been created by Zhu et al. (2001) and by Martzen et al. (1999). In these cases, the proteins are arrayed on the chip and the proteome can then be screened for interaction with particular substrates and ligands, and for certain classes of biochemical properties and activities. 

The most common type of capture molecules used on a protein chip are antibodies, though other proteins, such as peptides, nucleic acids, enzymes and receptors, have been spotted on to chips. Protein chips can be difficult to prepare, as the proteins need to be kept in an active state, in a high concentration and in a moist environment. Typical samples investigated are cell lysates for general research or blood and urine for diagnostic applications. Fluorescence detection methods are preferred since they are simple and sensitive. Other detection methods, where labelling is not required, can take the form of SELDl or AFM. 

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