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RIPA buffer recipe

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.

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Magnetic beads in Chemiluminescent immunoassay (CLIA)

How can magnetic beads improve CLIA tests?

The combination of CLIA and magnetic beads brings together all the advantages of both parts. CLIA is known for its high sensitivity which allows the detection of analytes at very low concentrations, and thus providing an excellent limits of detection in a wide dynamic range.

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Overview on Chemiluminescent immunoassay (CLIA)

General Introduction

Luminescence is the emission of light, and it can occur in many ways. In research and biomedical industry fluorescence and chemiluminescence are often used. Fluorescence is when light is absorbed then emitted by a substance. A photon of a higher energy state is absorbed, then a lower energy photon is emitted in another range of the electromagnetic spectrum. 

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Protein Molarity Calculator

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. 

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Magnetic immunoprecipitation (IP) input into western blot analysis

Immunoprecipitation

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.

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Monoclonal vs. Polyclonal antibodies

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.

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ELISA standard curve

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. 

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Acetate Buffer

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.

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GST fusion protein

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

 

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Cell based assay

Why do a cell based assay?

Cell based assays are used in research and industry as an in vitro test before going to an in vivo model. For example, a company that has developed a drug has to ask several questions about how it works before even moving to any live model. The company needs to know how well their drug binds either a surface receptor of interest or many they are trying to get the drug to go into the cell. Maybe the company wants to know how well the binding of their drug to a cell triggers the desired reactions inside the cell. These types of questions refer to the potency of a drug, or the mechanism of action. Cell based assays are an ideal way to directly test how a compound or protein is interacting with a cell before trying to figure out those molecules interact with an organism as a whole.

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