Blog

 

Cell Lysis buffer

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, 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.

Read More
 

Elution Buffer

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.

Read More
 

Ferrimagnetic materials

We are all familiar with magnetic materials, but not so much with what exactly ‘magnetic’ means. The magnetic moment, usually quoted as ‘m’, it is defined as the property that makes a material  align with an external magnetic field.

Read More
 

Indirect and Direct Elisa

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.

Read More
 

Direct and Indirect Elisa protocol

As mentioned previously in the discussion about indirect and direct elisa, the antigen is applied to the surface of the elisa plate whereas in a sandwich elisa a capture antibody is attached directly to the surface of the plate. Aside from this major difference the indirect and elisa protocol is very similar to the sandwich elisa protocol. There are plenty of blocking and washing steps to avoid non-specific binding, and there are incubation times to allow antibodies and antigens to bind properly.

Read More
 

Protein assay

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 specific shapes to perform specialized functions within the cells and tissues of all living organisms. It is no wonder that the ability to analyze proteins with a protein assay is fundamental to biological research and clinical diagnosis. The purpose of the protein assay is to determine the amount or concentration of protein in a sample. This is an important step before fancier labeling techniques such as biotin labeling or conjugation to enzymes or magnetic nanoparticles. It is important to know the concentration of proteins that you are working with before attempting to modify them in order to enable accurate and efficient labeling.

Read More
 

Immunoprecipitation protocol

Immunoprecipitation (IP) is a method used to purify target proteins from whole tissue or from cell culture. There are different types of IP: Single protein, Co-immunoprecipitation (co-ip), Chromatin immunoprecipitation(CHIP), RNA immunoprecipitation (RIP), and tagged protein immunoprecipitation. The basic steps of the immunoprecipitation protocol apply to all types of IP, but the goals of each assay are slightly different. The most basic method isolates a single target protein from the sample. Co-ip is used to pull out partner proteins and protein complexes. The CHIP assay is used to identify protein-DNA interactions within the histone. RIP is similar to CHIP except that it is protein-RNA interactions. Tagged protein immunoprecipitation is used when there isn't an ideal antibody candidate to bind to the target protein.

Read More
 

CHIP assay

A CHIP assay is short for Chromatin Immunoprecipitation assay. This technique is used to gain insight into the region of the genome a particular protein is associated with. To do this the CHIP assay captures information about the protein-DNA interactions in the histones of genomic DNA. The protein-DNA binding structure is preserved because the histone is crosslinked before the cell is lysed. The protein and DNA are bound together even after the chromatin is broken up into smaller pieces. Then the protein-DNA conjugates are captured by immunoprecipitationwith antibodies binding specifically to regions in the protein of interest. Then the DNA is extracted from the proteins and analyzed by targeted qPCR or genome-wide next-generation sequencing (NGS).

Read More
 

Sandwich ELISA

The sandwich ELISA is a type of Enzyme-linked immunosorbent Assay that uses two antibodies: a capture antibody and a detection antibody. The purpose of any ELISA is to detect the presence of a target antigen in a sample. In a sandwich ELISA the target antigen is bound between a capture antibody and a detection antibody. The capture antibody is immobilized on a surface. This is different from direct and indirect ELISAs where the antigen is immobilized onto the surface.

Read More
 

Biotinylated proteins and oligonucleotides

Biotinylation is the process of attaching a biotin tag to a molecule. The molecule can be a protein or an oligonucleotide. Biotinylated molecules are useful in many biological contexts, but are primarily used for capture or detection of target molecules. Biotinylated molecules are used in Western blotting, ELISA, flow cytometry, and other inventive detection methods.

Read More

Leave a comment