Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
PERIODIC ACID-SCHIFF Periodic acid–Schiff (PAS) is a staining method used to detect polysaccharides such as glycogen, and muco-substances- glycoproteins, glycolipids
The reaction of periodic acid oxidizes the vicinal diols in these sugars, usually breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or in the ring of the monosaccharide units that are parts of the long polysaccharides, and creating a pair of aldehydes at the two free tips of each broken monosaccharide ring.
The oxidation condition has to be sufficiently regulated so as to not oxidize the
aldehydes further. These aldehydes then react with the Schiff reagent to give a purple-magenta color. A suitable basic (Schiff) stain is often used as a counterstain. FLORESCENCE STAINING FLUORESCENT STAINING Oriole Gel Stain Easy-to-use, fast (90 min) and sensitive No destain or fixation required Compactable with downstream process and mass spectrometric analysis Nano gram sensitivity, no background Flamingo Gel Stain Two steps, time consuming (5hr) Compactable with mass spectrometric analysis and Edman-based sequencing To be used with Laser based scanners SYPRO Ruby Gel Stain Detection of glycoprotein, lipoproteins and metalloproteins No detection of nucleic acids in the sample ZYMOGRAPHY ZYMOGRAPHY Zymography is an electrophoretic method for studying/detection of enzymes or proteolytic activity.
Method is based on SDS gel impregnated with a protein substrate which is
degraded by the proteases resolved during the incubation period.
The technique is particularly useful for analyzing the proteinase composition
of complex biological samples.
Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background.
Zymography is also widely used to study various aspects of Matrix
Metalloproteinase (MMP) function. MATRIX METALLOPROTEINASE MMPs were discovered in 1962 -was first observed during tadpole tail development
It is capable of degrading all kinds of extracellular matrix proteins, also can
process a number of bioactive molecules
Physiological processes:- Embryogenesis, Angiogenesis, Activation of cell
When specific substrate is co-polymerized with the acrylamide
In zymography, the proteins are separated by electrophoresis under
denaturing conditions- SDS (sodium dodecyl sulfate) with non-reducing sample buffer
The separation occurs in a polyacrylamide gel containing a specific substrate
that is co-polymerized with the acrylamide SUBSTRATE ZYMOGRAPHY Gelatin Zymography– Gelatin zymography is mainly used for the detection of the Gelatinases, MMP-2 and MMP-9
Casein Zymography– suitable for the detection of MMP-1, MMP-7, MMP-12, and MMP- 13
Collagen Zymography– used for the detection of MMP-1 and MMP-13, but MMP-2 and MMP-9 can also be detected -The incorporation of native collagen fibers in polyacrylamide gels appears unsuitable for zymography because of their complicated structure, but SDS disrupts most of the fibrillar organization of the collagen, allowing proteins to run into the gel. SUBSTRATE ZYMOGRAPHY Heparin-Enhanced Substrate Zymography- It is known that the extraction of MMPs from tissue in the presence of heparin results in an enhancement of MMP activity.
The addition of heparin to the samples during or prior to electrophoresis also
enhances MMP activity.
Used for MMP-7
The mechanisms by which heparin seem to enhance
MMP-7activity in zymography are: (i) the induction of a conformational change (ii) the facilitation of refolding (iii) the reduction of autolysis (iv) the increase of anchorage of the MMP in the gel REVERSE ZYMOGRAPHY 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
TIMPs (Tissue Inhibitors of Metalloproteases) can be detected by reverse
zymography, which is a modification of zymography for MMPs
Besides gelatin, an MMP is also incorporated into the gel, usually MMP-2.
During the activation step after electrophoresis, the MMP-2 only digests the gelatin in areas where TIMPs are absent.
Thus, after staining, the gel will be colourless, except for the TIMP bands REVERSE ZYMOGRAPHY 2D-GEL ZYMOGRAPHY In situ ZYMOGRAPHY It is a unique laboratory technique that enables the localisation of matrix- degrading metalloproteinase (MMP) activity in histological sections.
Frozen sections are placed on glass slides coated with fluorescently labelled matrix proteins
After incubation MMP activity can be observed as black holes in the
fluorescent background due to proteolysis of the matrix protein.
This technique can be combined with immunohistochemistry to enable co-
location of proteins such as cell type markers or other proteins of interest.
Additionally, this technique can be adapted for use with cell cultures, permitting precise location of MMP activity within cells, ADVANTAGES OF ZYMOGRAPHY
Expensive materials are not required (e.g., antibodies)
Proteases with different molecular weights showing activity towards the same substrate can be detected and quantified on a single gel.
MMPs in solution are often associated with endogenous tissue inhibitors of
metalloproteases (TIMPs). During electrophoresis the inhibitors dissociate from the MMP and do not interfere with detection of the enzymatic activity.
On the basis of molecular weight markers, the molecular weight of the
proteolytic band can be determined, and by comparison with recombinant proteins and the use of specific protease inhibitors the type of protease can be established If you would like to donate us? Scan below and donate us 0.013$ (US dollar) (5Rs Indian rupee) Contact: If you want PPT/PDF files, please contact below. Email: gnccmysore@gmail.com Telegram:+919738137533(only for Chat)
2004 Simvastatin, An HMG-CoA Reductase Inhibitor, Reduced The Expression of Matrix Metalloproteinase-9 (Gelatinase B) in Osteoblastic Cells and HT1080 Fibrosarcoma Cells
2004 Simvastatin, An HMG-CoA Reductase Inhibitor, Reduced The Expression of Matrix Metalloproteinase-9 (Gelatinase B) in Osteoblastic Cells and HT1080 Fibrosarcoma Cells
