When doing a Western blot procedure, the macromolecules are first separated using gel electrophoresis. The separated macromolecules are transferred onto a second matrix (nitrocellulose or polyvinylidene difluoride (PVDF) membrane) and the membrane is blocked to prevent the nonspecific binding of antibodies to its surface. The transferred protein is then complexed with an enzyme-labeled antibody (probe) and an appropriate substrate is added to produce a detectable product.
The Protein Man's Blog | A Discussion of Protein Research
Tags: Protein Detection
In biochemistry, dialysis refers to the process of separating small, unwanted compounds from macromolecules in the sample solution through selective and passive diffusion. Basically, the sample and the buffer solution (dialysate) are placed on opposite sides of the membrane. Since dialysis works by diffusion, molecules will naturally move from areas of higher concentration to areas of lower concentration until the point where equilibrium is reached. This, in turn, will facilitate the separation of molecules in both the sample and the dialysate.
Tags: Sample Clean Up
Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state. HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique? To understand the mechanism behind this type of chromatography, here are some things you definitely need to know.
Tags: Basics of Hydrophobic
It is interesting to note that while detergent-solubilized membrane proteins can be purified and concentrated through phase separation (which is a simple, efficient and cost-effective method that can be used for this purpose), most membrane protein scientists do not use this method or even know that such methods exist. To better understand and appreciate how this method works, here are some things you definitely need to know.
Phase Separation: Understanding the Basics
In a nutshell, phase separation (also known as cloud point extraction) refers to the state wherein the micelles in an aqueous solution become immiscible with water and form large aggregates that will separate from the water phase. This condition is usually achieved upon the addition of detergents or when the temperature or salt concentration of the solution is altered.
Tags: Protein Purification
Researchers have traditionally used agarose beads for immunoprecipitation (IP). However, there has been a growing trend in recent years favoring the use of magnetic beads. According to a recent survey of 1,013 scientists, 60% of the respondents will start using magnetic technology in the next three years.
When it comes to Western blotting, choosing the right membrane for your application usually spells the difference between success and failure. However, this is usually easier said than done since you need to consider the properties of your protein and the downstream detection steps required in your application before you can determine which membrane can give you the results that you need.
Tags: Western Blotting
The 6xHis tag, also known as polyhistidine tag, His6 tag and/or hexa histidine tag, is an amino acid motif consisting of at least 6 histidine residues fused to the carboxyl (C-) or amino (N-) terminus of a target protein in transfected cells. This tag is most commonly used in the production of recombinant proteins since the string of histidine residues binds to several types of immobilized ions (such as nickel, cobalt and copper) under specific buffer conditions to allow for the simple detection and purification of His-tagged proteins.
Tags: Protein Purification
Agarose beads are small spherical beads of agarose gel which are commonly used in gel filtration or molecular size exclusion chromatography and biomolecular purification and immobilization. These beads act as porous gel to filter mixtures of molecules based on their individual sizes. And since these beads are easy to activate, they can also be used to bind biomolecules in a reversible or irreversible manner. In addition, their inert nature and unique internal surface area can also be activated for ligand attachment, making them the ideal basis for various affinity chromatography beads such as protein A and G, and glutathione.
Basically, spectrophotometry is one of the most widely used analytical procedures in biochemistry. It is commonly used to estimate the level of an analyte in solution and is ideal for simple routine determination of small quantities of materials. This method is based on the two laws of light absorption by solutions, namely Lambert's Law and Beer's Law.
Tags: Protein Estimation
Tagging your protein of interest can be extremely useful since it simplifies the purification protocol, improves the yield and solubility of your protein of interest and promotes the proper folding of their fusion partners. Due to their versatility, affinity tags (peptide sequences that are appended to the target protein) are recognized as one of the most powerful tools that can be used for basic biological research and in structural and functional proteomics as well.
Tags: Protein Purification