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Antibodies are powerful research tools used in various lab techniques. Here we provide a brief overview of the most popular lab techniques, highlighting how they use antibodies.
Updated May 11, 2022.
ELISA is a plate-based technique enabling the detection of antigens in biological samples. Like other immunoassays, ELISA relies on antibodies to detect a target antigen using highly specific antibody-antigen interactions. ELISA enables the quantification and characterization of analytes and molecular interactions.
In an ELISA, the antigen is immobilized to a solid surface either directly or more commonly via a capture antibody, itself immobilized to the surface (Fig. 1). The surface is washed, then incubated with detection antibodies conjugated to molecules such as enzymes or fluorophores.
In the antigen's presence, these detection antibodies will remain bound to the plate, providing a signal. The strength of this signal corresponds to antigen concentration within the sample.
Figure 1. Sandwich ELISA setup. A capture antibody on a multi-well plate will immobilize the antigen of interest. This antigen will be recognized and bound by a detection antibody conjugated to biotin and streptavidin-HRP.
An ELISA is typically performed in a multi-well plate (96- or 384-wells), and the analytes' immobilization facilitates the separation of the antigen from the rest of the sample components. These characteristics make ELISA one of the easiest assays to perform on multiple samples simultaneously.
There are four main types of ELISA: direct, indirect, sandwich, and competitive – each with unique advantages, disadvantages, and suitability. The most appropriate ELISA format for each experiment will depend on many factors, including desired sensitivity, specificity, and assay time. See more information here to help you choose the right type of ELISA.
For the same cost as a standard ELISA, our SimpleStep ELISA™ kits can halve assay time without compromising sensitivity or reliability.
Enzyme-linked immunospot (ELISPOT) is used to detect proteins secreted by cells, such as cytokines and growth factors. The technique enables quantification and comparison of immune responses to various stimuli.
Cells are grown in 96-well plates with antibody-coated PVDF or nitrocellulose membranes. The secreted proteins of interest are detected using primary and conjugated secondary antibodies. Cells secreting the protein of interest will appear as a spot of color or fluorescence. Membranes are scanned and analyzed to quantify the number or proportion of cells secreting the protein.
Western blot is widely used in research to separate and identify proteins. Western blot allows us to detect proteins, determine the relative protein levels between samples, and establish the target's molecular weight, providing insight into its post-translational processing.
Western blot involves three main steps: (1) separation of proteins by size, (2) transfer of proteins to a membrane, and (3) visualizing the target protein using primary and secondary antibodies (Fig. 2).
In the first step, the proteins are loaded onto a gel and separated based on size by gel electrophoresis. Protein bands are then migrated to a membrane using an electrical current. Protein transfer to the membrane is essential because gels used for electrophoresis provide an inferior surface for subsequent immunostaining, ie, antibodies don't stick to the gel's proteins.
Finally, the membrane can be further immunostained with antibodies specific to the target of interest and visualized using secondary antibodies and detection reagents.
Figure 2. A simplified diagram of western blotting.
Immunoprecipitation (IP) is a versatile technique that isolates and purifies individual and complexed proteins. In this technique, antibodies are immobilized on solid-phase substrates (eg, magnetic/agarose beads), capturing antigens from complex solutions.
Chromatin immunoprecipitation (ChIP) is used to determine whether a given protein binds to a specific DNA sequence in vivo. ChIP allows researchers to identify specific genes and sequences where a protein of interest binds across the entire genome, providing critical clues to their regulatory functions and mechanisms.
The ChIP procedure (Fig. 3) utilizes an antibody to immunoprecipitate a protein of interest, such as a transcription factor, along with its associated DNA. The associated DNA is then recovered and analyzed by PCR, microarray or sequencing to determine the genomic sequence and location where the protein was bound.