#StandardsGoals for 2024: What’s new for BISAC - Tech Forum 2024
Rise vibha investigation presentation (1)
1. +
Analysis and
Isolation of
Plasminogen
Activator from
Mammalian Cell
Culture Broth
Delaine Zayas-Bazán Burgos
John Muñoz Torres
Vibha Bansal PhD
Mr. Javier Rosado
Mr. Carlos Castrodad
Mrs. Natalia Espada
3. +
Introduction
“Magnetic nanoparticles continue to garner
widespread interest in biomedical
applications,such as visualization agents in
MRI, therapeutic vehicles for drug delivery
and heat mediators in hyperthermia.”
Reynolds and Hilt (2010).
Mihaiescu and his team (2012) worked
with nanoparticles as therapeutic vehicles
for drug delivery in E.coli
13. +
Separates proteins accordin
g to their electrophoretic
mobility and no other
physical feature.
SDS is a detergent applied
to protein sample to impart
a negative charge to all of
proteins.
In most proteins, the binding of
SDS to the polypeptide chain
imparts an even distribution of
charge
This causes a separation by
approximate size during
electrophoresis.
Based on SDS-PAGE
Includes a substrate co-
polymerized with the gel
Utilized for the detection
of enzyme activity.
Samples are prepared in the
standard SDS-PAGE treatment buffer
WITHOUT a reducing agent or boiling
in order for the enzyme to retain its
native state
It is incubated at 37°C to leave the
enzymes the correct environment for
them to digest the protein or substrate.
This causes white bands, in which the
protein has been digested.
SDS-Page Zymography
15. +
• SDS-Page
• Zymography
Polymerize
• Load and run the
gel
• Leave overnight at
room temperature
in fixative
SDS-Page
• Load and run the
gel at 4°C
• Stain with Coomasie
Blue
• Incubate overnight
at 37°C
• Destain
Zymo
• Take pictures
• Perform Protein
Estimation
• Perform Enzyme
Activity
Analysis
17. +
Results
These results shows
that the protein of
interest, PA, was
successfully isolated
from the bulk of
proteins available
They also provide an
ascertain that is our
protein, and not any
other protein.
HeLa
Load
HeLa
Spent
Eluate
Plasminogen
Activator
Zymography with
Coomassie Blue
Staining
20. +
Conclusions
The experiment was successful
Plasminogen Activator was isolated and
analyzed.
The protein was isolated at a fold
purification of 10
The fold purification is a measure of how much
more pure your protein is after a purification step
in comparison to the crude.
21. +
Further Works
This experiment can be utilized in further
analysis of the protein Plasminogen
Activator
The experiment can also be used as a base
for further experimentations about protein
separation with treated magenetic
nanoparticles
22. +
References
Balaure P, Buteica A, Grumezescu A, Mihaiescu D, Mihaiescu O,
Mogosanu D,Trăistaru V,Vasile B. 2012.Bioassay and
Electrochemical Evaluation Of Controlled Release Behavior Of
Cephalosporins From Magnetic NanoparticlesDigest Journal of
Nanomaterials and Biostructures. [Internet]; [Revised 2012
February 24, Cited 2013 May 16] 7(1). <a
href="http://search.ebscohost.com/login.aspx?direct=true&db=a9
h&AN=74616350&site=ehost-live">BIOASSAY AND
ELECTROCHEMICAL EVALUATION OF CONTROLLED RELEASE
BEHAVIOR OF CEPHALOSPORINS FROM MAGNETIC
NANOPARTICLES.</a
Eisenhour David, Hickman Jr. Cleveland, I’Anson Helen, Keen Susan
L, Larson Allan, Roberts Larry S. 2008. Integrated Principals of
Zoology. Fourteenth Edition. United States of America. McGraw-Hill.
23. +
References
Erf Gisela F, Kanayeva Damira A, Li Yanbin, Rhoads Douglas, Slavik
Michael F, Tung Steve, Wang Ronghui. 2012. Efficient Separation and
Sensitive Detection of Listeria monocytogenes Using an Impedance
Immunosensor Based on Magnetic Nanoparticles, a Microfluidic Chip, and
an Interdigitated Microelectrode. Journal of Food Protection. [Internet];
[Revised 2012 November; Cited 2013 May 11] 75(11).
http://search.proquest.com.uprcdb.cayey.upr.edu:2048/docview/11155922
25?accountid=44829
Frimpong Reynolds A, Hilt J Zach. 2010.Magnetic nanoparticles in
biomedicine: synthesis, functionalization and applications. Magnetic
nanoparticles in biomedicine: synthesis, functionalization and applications.
Future Medicine Ltd [Internet]; [Revised 2010 November; Cited 2013 May
11] 5(9) DOI:
http://dx.doi.org.uprcdb.cayey.upr.edu:2048/10.2217/nnm.10.114
http://search.proquest.com.uprcdb.cayey.upr.edu:2048/docview/81585852
9?accountid=44829