The collection of tissue is a fundamental procedure for research and clinical biology. Before collection it is important to consider which method will be used to preserve the tissue and prepare it for histology or molecular analysis. There are two options to choose from when collecting and preserving tissue: frozen or formalin-fixed paraffin-embedded (ffpe). Each has its strengths and weaknesses, but these are only apparent when the intended use of the tissue is considered.
The objective of magnetic bead cell isolation is to enrich a specific cell type from a mixed population. The technique is also be used to isolate proteins, DNA, and RNA for further research or therapeutic purposes. The versatility and specificity of magnetic bead cell isolation is made possible by functionalized bead surfaces that specifically recognize a target molecule. Magnetic beads are composed of a ferrous iron-oxide core surrounded by a polymer shell, or a magnetic ‘pigment’ embedded in a polymer matrix.
Anti human IgG is the most prevalent antibody isotype in human serum. It plays a critical role in the adaptive immune response. When a pathogen invades a human body it is intercepted by a white blood cell called a B-cell. These B-cell lymphocytes have cell surface receptors that bind a wide array of pathogens. Once activated, the B cell divides and produces offspring cells which secrete very specific antibodies capable of identifying that exact invading pathogen. These antibodies recognize a specific antigen binding site.
A GST (Glutathione-S-transferase) fusion protein is useful for affinity chromatography and immunoprecipitation. The natural form of GST is an enzyme that catalyzes the protective mechanisms of glutathione. Glutathione is an antioxidant that prevents cell damage by reactive oxygen species. However, the GST fusion protein is not natural. It is a genetically engineered protein that has become a useful biotechnological tool.
Immunoprecipitation, ELISA, antibody purification, and multiplex assays require the attachment of specific antibodies to a solid support such as a column, polystyrene plate, agarose bead, or superparamagnetic nanoparticle. There are a number of ways that antibodies are attached to solid supports. Some of these include:
- covalently bonding the antibody’s primary amines directly to the surface
- biotin-streptavidin affinity linkages
- protein A and G
Biotinylation means attaching a biotin tag to a molecule. The purpose of biotinylation is to create a controlled site for biotin-streptavidin affinity binding. Biotin and streptavidin have an extraordinary affinity for each other (Kd=10^-15). This affinity is resistant to changes in temperature, pH, and salt concentration and is extremely specific. These properties make biotinylation a useful tool for engineers who are developing new purification and detection methods. A commercially available biotinylation kit makes the process even easier.
Antibodies are an important part of the immune system. When the body is infected with an antigen, the immune system generates an antibody specific to that antigen. Biotechnology capitalizes upon this natural immune process. Antibodies are used in many research applications as well as in immunoassays for disease detection. We use the specificity of the antigen/antibody binding for immunoprecipitation and ELISA assays. We use flurophore-conjugated antibodies for labeling molecular targets on individual cells and whole tissue. We use antibody purification to obtain antibodies for biosensors to detect disease. These antibodies, depending on the application, are commonly obtained by antibody purification from humans, rats, rabbits, mice, and chicken.
The synthesis of complementary DNA (cDNA) is fundamental to the study of RNA expression in cells and tissues. RNA is fragile and not compatible with the polymerase chain reaction (PCR). Therefore, an intermediary step is needed to convert RNA to cDNA for analysis by quantitative PCR (qPCR). This qPCR technique utilizes specific primers and polymerase enzyme to amplify targeted sequences of cDNA. Since the cDNA is complementary to the initial mRNA isolate, this technique provides a quantitative readout of mRNA levels in the sample. The need for cDNA is so great that many companies sell cDNA synthesis kits to simplify the procedure.
Co-immunoprecipitation is a protein extraction technique that specifically targets protein-protein interactions. It is slightly different from immunoprecipitation. Immunoprecipitation utilizes antibodies immobilized on a mobile support to capture target proteins. Co-ip takes this concept one step further by using antibodies to target not the direct antigen that binds to the antibody, but any protein that binds to the antigen and is pulled out with it. This makes co-ip an ideal technique for studying protein complexes. The main concern when developing a co-ip protocol is to ensure that the lysis, wash, and elution buffers do not denature the proteins. Otherwise the tertiary structure of the proteins will deteriorate and the protein-protein interaction may be altered or completely lost.