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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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Virology test

Introduction to virology and virology tests

Virologists are scientists that study viruses, which are found in astounding numbers on earth (1031!). Viruses survive only in the cells of organisms they infect, making them a parasite. Whether or not they are “living things” is a philosophical argument for a different article. One of the major reasons for studying viruses is to understand the diseases they cause in humans. Virology research has led to the development of many virology tests. It is important to diagnose as well as monitor the progression of disease of certain viruses. This means that virology tests need to be available in clinics as well as in point-of-care form to test people in areas with fewer medical resources or infrastructure. Proper diagnosis can ensure that the spread of virus can be contained and treatment can be given to those in need. A current example of a crucial virology test is SARS-CoV-2 virology tests that detect the presence of viral nucleic acid or antibodies from human samples.

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Rna purification kit

Brief introduction to the importance of RNA

RNA (ribonucleic acids) are important macromolecules that come in many varieties and play more roles across all living organisms. Some predominant RNA are rRNA, tRNA and mRNA. tRNA is part of the process of translating proteins, rRNA is ribosomal RNA, and mRNA is messenger RNA which carries transcription information. RNA biology is an ever-growing field of research. RNA has even become a popular tool, such as the mRNA COVID-19 vaccine or CRISPR technologies. Researchers are even finding more non-coding RNA with important biological functions.

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Macs cell sorting: technology and advantages

Quick Background on Cell Sorting/Cell Isolation

Researchers use magnetic activated cell isolation also known as macs cell sorting or macs cell separation to enrich a specific cell type from a mixed population. Scientists or companies sort or isolate cells so they can study or grow colonies of a single type of cell. They may use that type of cell for a specific type of functional assay crucial to that cell type or they might be interested in stem cells. Many labs use flow cytometry, also known as FACS (fluorescence activated cell sorting). This is a specialized and expensive technique that often resides in a core facility at a research institution. MACS has emerged as a cheaper alternative for cell sorting. Both these technologies use the highly specific antibody-antigen interaction to probe cells by their surface antigens by their specific antibody. Magnetic bead cell isolation has a more simple protocol and components than FACS, the process is briefly described below. 

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mRNA isolation

Introduction and inspiration of mRNA

There is a lot to learn from the mRNA (messenger RNA) that is present in cells, which makes up around 5% of the total RNA in cells. The presence of certain mRNA sequences can inform us about what proteins are most likely being translated at the moment so that biological processes can take place. mRNA has also emerged as a top vaccine for the novel coronavirus of 2019, also known as SARS-CoV-2. How can a messenger RNA be a vaccine you ask? mRNA works as a vaccine by providing our cells with the sequence to make the major protein found on SARS-CoV-2 called “spike” protein. Once our cells have made spike protein, our body will launch an immune response against it, and immune cells will make antibodies against the spike protein. To use and study mRNA, you must first perform mRNA isolation, which we will introduce in this article. 

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