Zymography is a technique in biochemistry that uses electrophoresis to disperse particles. A substrate is used to react with an enzyme in a polycrylamide gel, which is known as SDS-PAGE. The gel is kept intact and stained to identify enzyme digestion. The process is used to identify the properties of proteins and enzymes for genetic research, pharmaceutical, and medicinal applications. Safety precautions must be taken as the materials used are toxic. Some enzymes may not respond as expected, causing problems with results.
Zymography is a technique performed by biochemists in which particles are dispersed using an electrical charge in a process known as electrophoresis. Specifically, the process uses a substrate in which an enzyme will react by polymerization with a polycrylamide gel, a water-based solution composed of toxic materials. This zymogram gel is known as sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).
A biochemist performing zymography will prepare the zymogram gel using the electrophoresis method. Usually this method is done in combination with boiling or gel reduction. However, in zymography, the SDS-PAGE gel is kept intact. After this step, the SDS portion of the compound is removed using a material known as Triton X-100, an oxidizing cleaner from the BASF company. This material is held at 98°F (about 37°C) for a period of time in an advanced incubator. A biochemist will then stain the remaining material using a black or blue compound. This allows a researcher to identify where and how digestion by enzymes occurs in the substance.
While SDS-PAGE gel is the substance most commonly used in zymography, other types of material can be used to identify the digestion rate and capacity of an enzyme. Many times this includes adding a starch film to aid the visual identification process.
Biochemists and biomedical engineers commonly use this process in an attempt to learn about new forms of enzymes. By defining the digestion rate and movement patterns of an enzyme, the different properties of human and animal proteins can be identified. This is used for some genetic research and analysis, as well as pharmaceutical and medicinal applications.
The zymography process must be handled with care. While it can mostly be done with minimal safety precautions, any scientist or researcher handling the materials should make sure to avoid physical contact. Both Triton X-100 Cleaner and SDS-PAGE Gel can be highly toxic to skin.
There are a number of enzymes that do not respond as expected to this process. For example, the digestive glycosidases of D. melanogaster can survive reduction processes in other types of digestive examination. It will also survive the heating process up to 122°F (approximately 50°C), well beyond the level needed in the traditional zymography process. When an enzyme survives the process without fully digesting the SDS-PAGE gel, increased band resolution is observed in the final stages. This can cause problems with getting correct results from the zymography process.
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