Zymography
Encyclopedia
Zymography is an electrophoretic technique, based on SDS-PAGE
, that includes a substrate
co-polymerized with the polyacrylamide
gel, for the detection of enzyme
activity.
or casein
) is embedded in the resolving gel during preparation of the acrylamide gel. Following electrophoresis
, the SDS
is removed from the gel (or zymogram) by incubation in unbuffered Triton X-100, followed by incubation in an appropriate digestion buffer, for an optimized length of time at 37°C. The zymogram is subsequently stained (commonly with Amido Black or Coomassie Brilliant Blue), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme.
The standard protocol may require modifications depending on the sample enzyme; for instance, D. melanogaster digestive glycosidases generally survive reducing conditions (i.e. the presence of 2-mercaptoethanol or DTT
), and to an extent, heating. Indeed, the separations following heating to 50°C tend to exhibit a substantial increase in band resolution, without appreciable loss of activity.
A common protocol used in the past for zymography of α-amylase activity was the so-called starch film protocol of W.W. Doane. Here a native PAGE gel was run to separate the proteins in a homogenate. Subsequently, a thin gel with starch dissolved (or more properly, suspended) in it was overlaid for a period of time on top of the original gel. The starch was then stained with Lugol's iodine.
Reverse zymography copolymerizes both the substrate and the enzyme with the acrylamide, and is useful for the demonstration of enzyme inhibitor activity. Following staining, areas of inhibition are visualized as dark bands against a clear (or lightly stained) background.
In imprint technique, the enzyme is separated by native gel electrophoresis and the gel is laid on top of a substrate treated agarose
.
Zymography can also be applied to other types of enzymes, including xylanase
s, lipases and chitinases.
SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
, that includes a substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
co-polymerized with the polyacrylamide
Polyacrylamide
Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...
gel, for the detection of enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
activity.
Procedure
Samples are prepared in the standard SDS-PAGE treatment buffer, but without a reducing agent or boiling in order for the enzyme to retain its native state (and therefore its proteolytic activity). A suitable substrate (commonly gelatinGelatin
Gelatin is a translucent, colorless, brittle , flavorless solid substance, derived from the collagen inside animals' skin and bones. It is commonly used as a gelling agent in food, pharmaceuticals, photography, and cosmetic manufacturing. Substances containing gelatin or functioning in a similar...
or casein
Casein
Casein is the name for a family of related phosphoprotein proteins . These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65% of the proteins in human milk....
) is embedded in the resolving gel during preparation of the acrylamide gel. Following electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
, the SDS
SDS
-Science:* Safety data sheet or material safety data sheet, a form with data regarding the properties of a particular substance* Satellite Data System, a system of United States military communications satellites....
is removed from the gel (or zymogram) by incubation in unbuffered Triton X-100, followed by incubation in an appropriate digestion buffer, for an optimized length of time at 37°C. The zymogram is subsequently stained (commonly with Amido Black or Coomassie Brilliant Blue), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme.
The standard protocol may require modifications depending on the sample enzyme; for instance, D. melanogaster digestive glycosidases generally survive reducing conditions (i.e. the presence of 2-mercaptoethanol or DTT
Dithiothreitol
Dithiothreitol is the common name for a small-molecule redox reagent known as Cleland's reagent. DTT's formula is C4H10O2S2 and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide-bonded 6-membered ring . Its name derives from the four-carbon...
), and to an extent, heating. Indeed, the separations following heating to 50°C tend to exhibit a substantial increase in band resolution, without appreciable loss of activity.
A common protocol used in the past for zymography of α-amylase activity was the so-called starch film protocol of W.W. Doane. Here a native PAGE gel was run to separate the proteins in a homogenate. Subsequently, a thin gel with starch dissolved (or more properly, suspended) in it was overlaid for a period of time on top of the original gel. The starch was then stained with Lugol's iodine.
Other uses
In imprint technique, the enzyme is separated by native gel electrophoresis and the gel is laid on top of a substrate treated agarose
Agar
Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
.
Zymography can also be applied to other types of enzymes, including xylanase
Xylanase
Xylanase is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls....
s, lipases and chitinases.