setting of gel electrophoresis

2. Matrix Metalloproteinase (MMP)
Matrix Metalloproteinases (MMPs), a family of zinc-containing enzymes,
show the function of decomposing Extracellular Matrix (ECM) and participate
in the physiological processes of cell migration, growth, inflammation, and
metabolism.
Clinical and experimental studies have indicated that MMPs play an essential
role in tissue injury and repair as well as tumor diagnosis, metastasis, and
prognosis.
Currently, there are 23 MMPs identified in humans and 24 in vertebrates
starting from MMP-1 to MMP-28 excluding MMP-4, -5, -6, and -22.
A level of MMP regulation is performed by Tissue inhibitors of
metalloproteinases (TIMPs), a family of four proteins (TIMP-1 to -4) that bind
to the MMP catalytic site and regulate proteolytic activity.

3. MMP Structure and Types
 They consist of several
domains; pro-peptide,
catalytic, linker peptide and
the hemopexin (Hpx)
domains.
 MMPs subfamilies possess:
collagenases, gelatinases,
stromelysins, matrilysins,
and membrane-type MMPs
(MT-MMP).

4. MMPs In Cancer
MMPs are crucial in biochemical interplay between tumor and stroma.
Stromal cells produce the majority of MMPs in the tumour microenvironment,
bringing about ECM cleavage, thereby forming a path for cell movement from the
tumor niche into adjoining areas.
Interaction of tumor cells with neighboring stromal cells is critical in facilitating
cancer initiation and progression.
Tumor cells secrete growth factors which stimulate surrounding cells in the tumor
tissues to release MMPs, allowing tumor cells to migrate.

5. Zymography
Zymography is a technique to assess the enzymatic activity of protein.
The enzyme converts the substrate into a product which is detected by different
staining methods.
One of the most popular method is by separating the protein mixture by
polyacrylamide gel electrophoresis in which a substrate is incorporated within the
polyacrylamide gels.
These protein substrates present in the gel are degraded by the activated proteases
present in the sample after incubation.

6. Zymography (Cont.)
Staining the gels with coomasie blue shows the
proteolytically cleared sites as white clear bands
on a dark blue backgrounds.
Different substrates are added to the gels for the
detection of different MMPs, for example, gelatin
for MMP-2, and -9, casein for MMP-3, -10, and -
7.
MMPs remain inactive while they are with their
pro-domains and they need processing to get
activated.
SDS present in SDS-PAGE activates it by
denaturing the MMPs.

7. Western Blotting
Western blot is often used in research to separate and identify proteins from a
complex mixture of proteins extracted from cells.
The technique uses three elements to accomplish this task: (1) separation of
proteins by size (molecular weight), (2) transfer of the proteins to a solid
support, and (3) marking the target protein using a proper primary and
secondary antibody to visualize.
The unbound antibody is washed off leaving only the bound antibody to the
protein of interest.
The bound antibodies are then detected by developing the film.

8. Western Blotting (Cont.)
As the antibodies only bind to the
protein of interest, only one band
should be visible.
The thickness of the band
corresponds to the amount of
protein present; thus doing a
standard can indicate the amount
of protein present.
p-AKT
β-Actin