Proteins differ from each other in their size, molecular structure and physicochemical properties. These differences allow for protein analysis and characterization by separation and identification.
There are three major protein analysis techniques: protein separation, western blotting and protein identification.
- PROTEIN SEPARATION
- WESTERN BLOTTING
- PROTEIN IDENTIFICATION
- LIGHT SCATTERING
- MULTI-DETECTION GPC/SEC
- Circular Dichroism Spectrometry
- Isothermal Titration Calorimetry
Recombinant protein expression technology enables study of gene regulation and protein structure and function. Utilization of recombinant protein expression can also vary widely, from investigation of function in vivo to large-scale production for biotherapeutic drug discovery and structural studies. Using the right protein expression system for your specific application is critical to success. Consider protein solubility, functionality, purification speed, and yield when choosing an expression system. Proteins are typically separated by electrophoresis, where they are differentiated by size or mass, and isoelectric focusing, where proteins are separated by charge. Protein identification by mass spectrometry involves ionization of molecules to determine their mass-to-charge ratio. Identification can also be done via amino acid sequencing by Edman degradation, crystal imaging and surface plasmon resonance. With so many options available, laboratories typically use multiple strategies for protein analysis and characterization.
Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. It has concluded that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.
A technique called mass spectrometry permits scientists to sequence the amino acids in a protein. After a sequence is known, comparing its amino acid sequence with databases allows scientists to discover if there are related proteins whose function is already known. Often similar amino acid sequences will have similar functions within a cell. The amino acid sequence also allows scientists to predict the charge of the molecule, its size, and its probable three-dimensional structure. The charge and size can later be confirmed experimentally (via SDS-PAGE and double-dimension gels). To deduce the intricacies of three-dimensional structure, scientists will try to crystallize the protein to confirm its molecular structure through X-ray crystallography and/or nuclear magnetic resonance spectroscopy (pNMR).
The results from different kinds of studies have fundamental relevance, from the basic understanding of normal cell function, such as cell differentiation, growth, and division, to informing radically new approaches for treating disease. In fact, some human diseases can arise simply from a defect in a protein’s three-dimensional structure. Through the study of gene expression and proteins, it is easy to see how minute changes at the molecular level have a reverberating impact.