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 northern blot is a technique used to study gene expression via mRNA transcripts. The northern blot was named after the southern blot, which was developed to study DNA. The two techniques are the same except that the northern blot is used to detect RNA while the southern blot is used to detect DNA. The northern blot protocol, in brief, involves gel electrophoresis to separate mRNA by size, a blotting step to transfer the separated mRNA to a membrane, and a probe hybridization step to identify the mRNA sequence of interest. Even with the advent of powerful RNA analysis techniques such as RT-qPCR and sequencing, the northern blot is still useful for comparing gene expression between samples. The northern blot protocol is relatively inexpensive, and makes it easy to visualize the results on a single membrane.
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.
A cell lysis buffer is a critical first component to any isolation protocol. It is fundamental to the first step of protein or nucleic acid extraction as it aids in the chemical breakdown of cell membranes and compartments, enabling target molecules to escape. There are many types of lysis buffers; most are easy to make, but most are also commercially available. They are often included in kits for immunoprecipitation, co-ip, nucleic acid isolation, and others. When using a lysis buffer for protein capture it is a good idea to add protease inhibitors prior to use in order to protect proteins.
An elution buffer plays an essential role in every immunoprecipitation protocol or assay that requires the release of a target antigen from a capture antibody. Elution buffers are necessary in protocols utilizing a stationary affinity column, and are also required in protocols using mobile solid supports in solution.
A couple of months ago we described the sandwich elisa. Here we will discuss the other two main types of elisas—indirect and direct. Elisa is an acronym for enzyme-linked immunosorbant assay. The purpose of any elisa is to detect the presence of an antigen or antibody of interest. The indirect and direct elisa differ from the sandwich elisa because the antigen of interest is bound directly to the plate rather than a capture antibody.
As mentioned previously in the discussion about indirect and direct elisa, the antigen is applied to the surface of the elisa plate whereas in a sandwich elisa a capture antibody is attached directly to the surface of the plate. Aside from this major difference the indirect and elisa protocol is very similar to the sandwich elisa protocol. There are plenty of blocking and washing steps to avoid non-specific binding, and there are incubation times to allow antibodies and antigens to bind properly.