There are a number of staining methods that can be used to detect highly glycosylated proteins on SDS gels, even at very low levels (i.e. up to a few nanograms). Some of the most commonly used stains for this purpose include the Coomassie brilliant blue stain (for proteins with limited glycosylation) and silver stain (used in cases where high sensitivity is required).
The Protein Man's Blog | A Discussion of Protein Research
Tags: Protein Detection
Most people learn more by doing things rather than by just reading, watching or hearing about it. As such, providing hands-on learning can have a profound effect on learning in schools, particularly on science teaching.
While proteolytic enzymes such as proteases and phosphatases play an important role in living cells and help ensure the survival of the organism, the mechanisms that regulate the tightly controlled cellular environment is disrupted during cell lysis. When this happens, these enzymes may start cleaving a variety of proteins that they would otherwise not touch in intact cells. This situation often leads to reduced recovery of total protein and biologically meaningless representation of protein activities.
Proteases are enzymes that facilitate proteolysis, or the breakdown of protein and peptide molecules into smaller polypeptides and/or amino acids. These enzymes do their job by cleaving the peptide bonds linking the amino acids together in the polypeptide chains.
Tags: Protein Estimation
Dialysis is a solution-based separation technique used to facilitate the removal of small, unwanted compounds from macromolecules such as proteins, DNA, or polysaccharides through a semi-permeable membrane using the principle of selective diffusion. Although there are a lot of factors that affect the dialysis rate, the semi-permeable membrane is the single most important factor that can determine the success of the process.
After the protein bands have been separated by electrophoresis, researchers perform a dye-binding or color-producing chemical reaction to visualize the proteins embedded within the pores of the polyacrylamide gel matrix. Some of the most popular in-gel detection methods include staining with Coomassie dye, metallic silver, and zinc.
The bicinchroninic acid (BCA) assay, also known as the Smith assay, is a biochemical assay used to determine the total concentration of protein in a solution. Due to its ability to provide accurate determination of protein concentration and its compatibility with most protein sample types, protein laboratories around the world prefer the BCA assay over any other detergent-compatible assays.
We recently reviewed the advantages of total protein membrane stains as a loading control for Western blotting and demonstrated how they were an improvement on routinely used housekeeping genes.
Tags: Western Blotting
Western blotting is a very popular and sensitive protein detection system that is routinely used for multiple areas of protein research. The sensitivity of this detection system continues to improve with increasingly sensitive enhanced chemiluminescence systems and the increasing popularity of IR (infra-red) fluorophores and IR imaging systems:
Due to its high sensitivity, wide dynamic range, and high signal-to-noise ratio, enhanced chemiluminescence or ECL is considered as one of the most popular detection methods for a variety of western blotting applications in most protein laboratories around the world. This method also proves to be very useful in the quantification of a wide variety of biological materials such as cells, proteins, RNA, DNA, and a host of other analytes.
Tags: Western Blotting