Loading controls serve as a vital method of verifying the results acquired through western blotting. Loading controls are antibodies that are used to detect proteins within samples. When western blots are used to determine the levels of protein expression in a sample, loading controls ensure that the results aren't due to loading or protein transfer errors. When using a loading control, the right type of control must first be identified.
The Protein Man's Blog | A Discussion of Protein Research
A high background is one of the most common quality control issues encountered during the process of western blotting. When a high background occurs, it can be very difficult to distinguish the relevant data from the irrelevant data. A uniform high background can be caused by numerous issues and may take some work to resolve -- but without resolving the situation, the blot may not be readable.
DNA denaturation is the process of breaking down the DNA molecule, generally for the purposes of comparison or sequencing. As with many laboratory techniques, there are a variety of ways to denature DNA -- and each of them tend to be better for specific applications. The top three methods of DNA denaturation are heat, NaOH treatment, and salt. Each of these methods will break the bonds between strands, but may do so with a greater degree of accuracy or lessened disruption.
After the preparation of DNA solutions, some of the enzymes involved may be impacted by the residues of the chemicals that have been used. This may include excess salt, SDS, or other inhibitory substances. In order to properly utilize the results of the DNA preparation, it is sometimes required to perform a drop dialysis. Ideally, the drop dialysis will be able to "wash" the enzymes in question, removing the residue and providing a better and more accurate result.
Poor protein transfer can lead to either a weak signal or a lack of signal being observed during the process of western blotting. Though poor protein transfer does occur fairly often, it's also commonly disregarded as a potential cause for a failure. Consequently, it's an issue that should be considered first when trying to determine the cause of a poor signal.
Lytic enzymes have a fairly prominent role in protein and DNA extraction. There are multiple enzymes that can be potentially used for lysis, depending on the applications and your lab's unique needs. Understanding lytic enzymes is essential to the process of successful protein extraction. Though physical lysis has been used in the past, it's no longer feasible -- and it's always important that lysis be completed with the proper lytic enzymes, to yield the most valuable results.
Proteins are highly heterogeneous, complex bio macromolecules consisting of one or more long chains of amino acids. Proteins or peptides fold up to form secondary and tertiary structures, and associate with other protein subunits to form quaternary structures. Proteins are structurally and functionally different from each other and require distinct surrounding environment for their stability and activity. Proteins are susceptible to degradation, denaturation and precipitation when taken out of their native environment.
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:
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.