Mn ps presentaion rise...
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PROTEIN SEPARATION USING
MAGNETIC NANOPARTICLES
Anthony Maldonado Castro
Félix Valles
Dr. Vibha Bansal
RISE Program
Proteins
Proteins- macromolecules that consist of up to 20
different amino acids
Play key functions in the living system such as
carrying oxygen and controlling the sugar levels in
the blood.
Isolation of proteins may have different purposes
such as catalyst usage, therapeutics, dietary
supplements and structure studies.
What did we use?
SDS PAGE
Fibrin Zymography
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.
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.
Fibrin Zymography vs. SDS PAGE
SDS PAGE- used to determine the prescence of
proteins
Fibrin Zymography- used to determine enzyme
activity
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.
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.
Fibrinolysis- breakdown of fibrin clot to
prevent thrombus formation
PA
I
tPA
Plasminogen PlasminFibrin clot
degradation
Isolate Plasminogen Activator (PA) from
mammalian cell culture broth.
Objectives:
Plasminogen Activator (PA) will be isolated from
mammalian cell culture broth.
Hypothesis:
Procedure
Suspension Equilibration Incubation
Regeneration Eluates Wash
Desalting AbsorbanceSDS and
Zymography
Preliminary Results
SDS PAGE – Protein estimation
SDS PAGE: Protein estimation
Sample ABS 562nm Abs - Blank Concentration Conc. Average
Load HeLa2.31 2.223 4.98654105 4.384253
Load HeLad1.773 1.686 3.781965007
Spent HeLa1.946 1.859 4.170031404 3.835801
Spent HeLad1.648 1.561 3.501570211
Eluate HeLa10.109 0.022 0.049349484 0.026918
Eluate HeLa1d0.089 0.002 0.004486317
Eluate HeLa20.095 0.008 0.017945267 0.020188
Eluate HeLa2d0.097 0.01 0.022431584
Eluate HeLa30.095 0.008 0.017945267 0.01794
Eluate HeLa3d0.673 0.586 1.314490803
SDS PAGE
Only the marker and the
HeLa Load can be
appreciated in the gel
image
Zymography - Enzyme Activity
Sample Abs405 Abs - Blank Activity Average Act.
Load HeLa 0.149 0.091 0.1075 161.25
Load HeLa d 0.182 0.124
Spent HeLa 0.158 0.1 0.0995 149.25
Spent HeLa d 0.157 0.099
Eluate 1 0.069 0.011 0.0115 17.25
Eluate 1 d 0.07 0.012
Eluate 2 0.056 -0.002
Eluate 2 d 0.055 -0.003
Eluate 3 0.062 0.004
Eluate 3d 0.061 0.003
The only eluate that shows activity is Eluate 1
Zymography Gel
Future Work
Another SDS and (possibily) a Zymography
Increase initial TPA concentration
Use silver staining instead of Coomassi Blue
Acknowledgements
Dr Vibha Bansal
RISE Program
Alexandra Rosado
Osvaldo Vega
PROTEIN SEPARATION USING
MAGNETIC NANOPARTICLES
Anthony Maldonado Castro
Félix Valles
Dr. Vibha Bansal
RISE Program