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.
Magnetic properties of nanoparticles are used for drug delivery, therapeutic treatment, contrast agents for MRI imaging, bioseparation, and in-vitro diagnostics. These nanometer-sized particles are superparamagnetic, a property resulting from their tiny size—only a few nanometers—a fraction of the width of a human hair (nanoparticles are approximately 1/1,000 thinner than human hair). Superparamagnetic nanoparticles are not magnetic when located in a zero magnetic field, but they quickly become magnetized when an external magnetic field is applied. When returned to a zero magnetic field they quickly revert to a non-magnetized state. Superparamagnatism is one of the most important properties of nanoparticles used for biomagnetic separation.
Magnetic beads and separators have grown in popularity as a biotechnological tool over the past few decades. As we gained experience with the technology, we learned that all magnetic separators are not created equal. It turns out that it isn’t just the magnitude of the magnetic force that improves separation time, it is the way it spreads out over the working area that matters the most. A homogeneous magnetic separator is designed to ensure that every point within the working volume experiences the same magnetic force. This is especially important when scaling up a biomagnetic separation process from milliliters to liters.
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.
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.
Protein A beads like ELISA, Immunoprecipitation, 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
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.
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 protocol, 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.
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. In either case, the key component is an enzyme-linked detection antibody. The enzyme is either colorimetric or chemiluminiscent. Chemiluminiscent enzymes are popular because they are easily read by a luminometer plate reader, making the process easy and highly quantitative.