HRP stands for horseradish peroxidase, an enzyme derived from horseradish. Streptavidin is a protein derived from a species of bacteria in the genus streptomyces. Streptavidin has a high affinity for the molecule biotin. Streptavidin HRP is a streptavidin protein conjugated to HRP. HRP is used for detection/read out signals in an assay such as ELISA. Depending on which substrate you give the HRP, the enzyme will produce a different signal that you can read out with whatever hardware/technology you have in your laboratory. Let’s discuss the general protocol for using streptavidin HRP and the various substrates you can use to get an output.
Proteins are one of the four macromolecule building blocks of life. The other three are carbohydrates, lipids, and nucleic acids. Proteins are long strings of amino acids that fold together into what are called “hierarchical structures” in order to perform specialized functions within the cells and tissues of all living organisms. Let’s describe what the hierarchical structures for proteins are. First is the chain of amino acids that make up a protein, called its primary structure. Each amino acid has a “side chain” composed of elements that make that amino acid unique. The side chains interact with one another to create the next part of the hierarchical structure, the secondary structure. The secondary structure takes the form of an alpha helix or beta sheet. The next structure is the tertiary structure in which the sheets and helices fold in an physically and chemically favorable way to create a “folded” structure. When multiple folded proteins come together to form a bigger structure, that is called the quaternary structure.
Today we are going to talk about a piece of technology found in laboratories around the world, the spin column. It is used for “solid phase extraction.” In simple terms, the spin column has a solid material that can be used to retain or bind certain molecules while letting other molecules pass through it. When referring to spin columns, this usually refers to nucleic acid purification/isolation (DNA or RNA) or proteins. The starting material can vary from blood to tissue.
General introduction to RIPA
RIPA stands for Radio immunoprecipitation assay. Let’s start out by talking about what a radio immunoprecipitation assay is and why it is used. In general, an immunoprecipitation assay uses antibodies to pull a protein of interest. The “immuno” part refers to the antibody (a molecule of the immune system) and the “precipitation” refers to a substance coming out of solution. You can read about the immunoprecipitation protocol in our other article "immunoprecipitation protocol". The radio part of this refers to using isotope labeling to track molecules. In a RIPA assay you radiolabel an antigen so you can track it when it binds an antibody, while the antibody is used to precipitate the antigen.
Quick background on proteins
Proteins are a type of macromolecule made of amino acids. Each amino acid has an amino side chain, a carboxyl group side chain and between them are the atoms unique to each amino acid, typically these unique atoms are called the “side chain” or “r group.” There are 20 amino acids that make up the chains of proteins. There are a large number of combinations that can arise from 20 amino acids, their various placement in a chain, the length of the chain, and the secondary and tertiary structure of the chain. These many combinations give rise to the diversity of proteins and their functions in nature. There are two types of secondary structure that a protein chain can take, alpha helix and beta sheet. These secondary structures further take on a tertiary structure which can happen in many ways.
Immunoprecipitation (IP) is a technique for capturing specific proteins from a complex solution via antibody-antigen affinity. In an IP, the goal is to pull out a specific protein. For a co-IP, instead of identifying individual proteins, the goal of the technique is to identify protein complexes. The phrase “pulling down” protein is commonly used to explain the process, but this idea is somewhat dated now that magnetic nanoparticles have begun to replace traditional centrifuge-based methods. The protein capture efficiency can be measured by IP input into SDS page and western blot analysis.
Easy background on antibodies
Antibodies are proteins that act as part of our adaptive immune response. In response to a pathogen, our body immediately calls on immune cells that are part of our innate immune response. The next response is called the adaptive immune response, and during this process an immune cell called a B-cell generates antibodies. The primary job of antibodies is to bind to pathogens to neutralize their ability to infect cells and to act as a tag that signals the start of more immune mechanisms.
Background on ELISA
ELISA stands for enzyme-linked immunosorbant assay. To help you understand the usefulness of this technique we’ll start with a brief description of how it works. The first step is to immobilize a probe molecule to your ELISA plate, these plates are usually purchased through a vendor. A probe is a molecule that binds to a target (analyte) you are hoping to capture from a sample mixture. The probe binds to the bottom of the wells through passive adsorption to the plastic. You next add your sample and allow time for your target of interest to bind to the probe in the ELISA plate wells. Lastly, a secondary antibody is added to visualize where binding has occurred through a colorimetric or fluorescent signal. There are various versions of ELISA that modify the assay for what kind of molecule you are trying to detect in a sample and whether a primary detection antibody is available for your assay for example. You can discover which ELISA is best for you in these articles about direct and indirect ELISA, sandwich ELISA, competitive ELISA.
This blog post is going to be devoted to acetate buffer, a widely used buffer in laboratories and scientific industries. First let’s discuss acetate, a carboxylate which is the conjugate base of acetic acid (commonly known as the main component of vinegar). Acetate has a negatively charged oxygen which is why it becomes the salt, sodium acetate. In general, buffers vary in their composition because they range in their use from helping cells grow in a petri dish to stabilizing RNA for freezer conditions. Optimization of a buffer is crucial to ensure that the correct molecules are present and the pH works for the steps of your procedure in which the buffer is used.
In nature, the GST (Glutathione-S-transferase) protein is an enzyme that catalyzes the protective mechanisms of glutathione (GSH). Glutathione is an antioxidant that prevents cell damage by reactive oxygen species. A gst fusion protein is a protein that is tagged with GST protein. The fusion protein is made by adding the sequence encoding GST to the plasmid expressing your protein of interest. GST has an affinity for GSH making them a good pair for use in chromatography and immunoprecipitation