2. Proteins
Proteins- macromolecules that consist of up to
20 different amino acids
Play key functions in the living system such as
carrying oxygen, controlling the sugar levels in
the blood and defending against foreign
cells, pathogens and bacteria.
Isolation of proteins may have different
purposes such as catalyst
usage, therapeutics, dietary supplements and
structure studies.
4. Sodium dodecyl sulfate PAGE
An electrophoretic technique in which proteins
are separated according to size.
In this particular project it is used to estimate
the concentration of protein in each sample.
5. Fibrin Zymography.
Fibrin zymography is an electrophoretic
technique for the detection of hydrolytic
enzymes.
It can be used for peptidase investigation,
identifications and characterizations in
biological living systems.
For example, it could be used to detect low
levels or absence of thrombin, the active form
of prothrombin, which converts fibrinogen to
fibrin, that is essential for blood coagulation.
6. Fibrin Zymography vs. SDS
PAGE
SDS PAGE- used to determine the prescence
of proteins
Fibrin Zymography- used to determine
enzyme activity
7. Magnetic Nanoparticles (MNPs)
MNPs are more efficient than traditional
separation methods since they are:
Fast
Scalable
Easily automated and separated from other
suspended solids
They reduce the pretreatment and
chromatography stages into a single step
isolation when combined with affinity binding.
8. Tissue Plasminogen Activator
(tPA)
Tissue plasminogen activator (tPA), is a
protease found in endothelial cells involved in
the breakdown of blood clots by catalyzing
plasminogen to plasmin, an enzyme
responsible for fibrin clot breakdown.
“Since tPA is free of immune side effects and
has short half-life, it is considered an excellent
thrombolytic agent for medical use”. (Byong-
Gon P, et al. 2000)
9. Tissue Plasminogen Activator (tPA)
(cont.)
“t-PA is a poor plasminogen activator in the
absence of fibrin. However, in the presence of
fibrin, however, its activity is two orders of
magnitude higher. The kinetic model indicates
that both t-PA and plasminogen bind to fibrin in
a sequential and ordered way, yielding a cyclic
ternary complex in which t-PA has a markedly
enhanced affinity for its substrate
plasminogen.” (Collen D, Lijnen HR. 2009)
10. Fibrinolysis- breakdown of fibrin
clot to prevent thrombus
formation
α2-
antiplasmin
tP
A
Plasminoge
n
Plasmin
Fibrin clot
degradatio
n
11. Tissue Plasminogen Activator (tPA)
(cont.)
“Malignant tumors frequently secrete
plasminogen activator activity, and that their
malignancy correlates with the level of
“malignant protease” secreted. This protease
activity could be inhibited with plasma α2-
antiplasmin, a plasmin inhibitor that can
rapidly inactivate free plasmin in the blood.
(Collen D, Lijnen HR. 2009)
12. Tissue Plasminogen Activator (tPA)
(cont.)
“This study demonstrates that in this rat
thromboembolic model of stroke, tPA-induced
hemorrhage is dependent on blood pressure
and that pharmacological reduction of
hypertension during fibrinolysis can reduce the
risk of hemorrhagic transformation.” (Emiri T,
et al. 2001)
20. SDS PAGE
Only the marker and the
HeLa Load can be
appreciated in the gel
image. This might be
result of low
concentration of the rest
of the samples loaded
on to the gel.
21. SDS - PAGE
The second
SDS PAGE
was stained
using silver
stain. This time
elution 1 can
be easily
distinguished.
22. Conclusions
The use of magnetic nanoparticles is an
efficient method for protein separation.
Have an efficient reusability.
23. Acknowledgements
Dr Vibha Bansal
RISE Program
Alexandra Rosado
Osvaldo Vega
Natalia Espada
José J. Rosado
Mariana León