Carbohydrate affinity chromatography is a method of choice for purification of glycoproteins, lectins and other carbohydrate metabolite proteins. This affinity chromatography uses carbohydrate ligands such as carbohydrates, glycoproteins and carbohydrate matrices for purification. The matrix needs to be activated for covalent immobilization of the ligand. Various methods used for the coupling of ligands depending upon the activating reagents are as follows:
The Protein Man's Blog | A Discussion of Protein Research
Tags: Protein Purification
Gel filtration is a method of separating molecules by size. Among similar techniques, gel filtration is known as being a very simple and gentle procedure. When it comes to desalting and buffer exchange, gel filtration is often the preferred method as it can achieve better separation without any complex processes. Gel filtration is commonly used for chromatography, with the only negative being that it is very difficult to get high resolution results.
For the purposes of chromatography, fast flow agarose resin provides a stable, easy, and effective medium. Comprised of cross-linked agarose beads, fast flow agarose resin is able to improve upon both the speed and reproducibility of chromatography, offering good resolution and the ability to predictably scale up. Fast flow resins also have extremely good flow properties and superior loading capacity, making it ideal for faster separation cycles, better purification, and lessened dilution.
“Lectin” word originated from latin word “legree” which means “to select”. Lectins are proteins or glycoproteins that have at least one noncatalytic domain that binds specifically and reversibly to monosaccharides or oligosaccharides. Lectins whose sugar specificities are unknown are called hemagglutinins. Lectins are present in every organisms and are involved in biological functions such as adhesive, defence against pathogens, immunomodulatory and regulatory.
It's often necessary to determine the solubility of a chemical before you can begin working with it. Chemicals, especially those used in labs, are often concentrated and distilled, in order to be easier to use, transport, and store. But these highly concentrated chemicals will have to be properly reconstituted if they are to be used reliably. The solubility of a chemical is its ability to dissolve when presented with a certain solvent. When it comes to chemicals, it's often important to know how soluble the product is and which solvents are most appropriate for its applications.
There are a wide variety of ELISA substrates designed for different systems and detection methods. These ELISA substrates are designed to detect alkaline phosphatase (AP) and horseradish peroxidase (HRP). In addition to their sensitivity, other factors, such as cost and ease of use, may also factor in. Choosing the right ELISA substrates begins with determining the limits of detection required. From there, a general type of ELISA substrate can be selected and the specific substrate can be narrowed down. There are five major types of substrate available: PNPP, ABTS, OPD, TMB, and ONGP. Of these, TMB is one of the most versatile.
Cells are made up of recognizable structures that each serve their own purposes and have their own characteristics. Common subcellular markers are often used to distinguish cells from each other and to identify potential irregularities within the cellular structure. Though there are a multitude of subcellular markers available, a few of them are more common and more easily identified than others.
Extraction of proteins from cells and tissue of organisms is the first step towards isolation of proteins. The extracellular matrix needs to be removed or digested in case of tissue, the cell wall needs to be digested for organisms like bacteria, yeast and plants, and the cell membrane needs to be disrupted to release the proteins in solution. Traditionally, physical methods for disruption of cells and tissues are employed to release cellular proteins including sonication, french press, homogenization, manual grinding or using blenders. Although one is able to get the active proteins, these physical methods have several limitations:
Protein analysis and identification through mass spectrometry first requires a breakdown of each protein into their composite peptides. Once the protein has been broken down, the peptides can be separated through the use of a reverse phase column and the peptides and peptide fragments can be measured using a mass spectrometer.
Tags: Mass Spectrometry
Adjuvants play an important role in the antibody production by acting as immunopotentiators. They augment immune response via different mechanisms depending upon the adjuvant such as ‘depot’ effect, antigen presentation, antigen targeting, immune activation or modulation and cell-mediated response. The goal of adjuvant for antibody production is that high affinity, high titer and high avidity (for polyclonal) antibodies are raised. Both humoral and cell mediated response are necessary to achieve efficient antibody production. There are hundreds of preparations for adjuvants described in literature depending upon specific needs of an investigator. Nevertheless, most commonly and widely used adjuvants for antibody productions are few as listed below.
Tags: Antibody Production