The document describes an experiment to isolate plasminogen activator from a mammalian cell culture using PABA treated magnetic nanoparticles. Key steps included separating the protein using magnetic nanoparticles, analyzing the isolated protein through SDS-PAGE, zymography, and protein estimation assays. Results showed the successful isolation and purification of plasminogen activator from the cell culture broth.

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

2. +
Introduction
Proteins are macromolecules
composed of amino acids, joined by
peptide bonds.
Our purpose was to isolate
Plasminogen Activator from a
mammalian cell culture
 HeLa Broth

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

4. +
Introduction
What are nanoparticles?
 PABA Treated Magnetic Nanoparticles

5. +
Hypothesis
Plasminogen Activator can be
successfully isolated from a
mammalian cell culture utilizing PABA
treated Magnetic Nanoparticles

6. +
Work Plan
• MNP
• Chromatogram
Separation
• SDS Page
• Zymography
• Protein
Estimation
• Enzyme
Activity
Analysis

7. +
Separation
Separation
Image from Dr. Bansal’s
Handout

8. +
Materials
Reagents:
 HeLa Broth
 Binding Buffer
 Elution Buffer
 Regeneration Buffer
 PABA-Epoxy-IO
MNPs (50.0mg)
Equipment:
 Sonicator
 Rocker (120 rpm)
 UV
Spectrophotometer

9. +
Materials
 Seven (1.5mL) centrifuge
tubes
 One 15.0 mL centrifuge
tube
 One magnet
 Ice Bucket with ice
 Test Tube rack
 100-1,000 μL Micropipette
and tips
 Stop clock
 Beaker for waste collection
 Labeling Tape
 Marker
 Gloves

10. +
Analisis

11. +
Materials needed for SDS-Page
 Stacking gel 4.0%
 dH2O 2.795 ml
 Stacking buffer 1.25 ml
 Acrylamide 0.662 ml
 20% SDS 25.0 µl
 10% Glycerol 25.0 µl
 TEMED 6.25 µl
 APS (100 mg/ml) 25.0 µl
 Separation gel 10.0%
 dH2O 1.68 ml
 Separating buffer 1.67 ml
 Acrylamide 2.22 ml
 20% SDS 50.0 µl
 10% Glycerol 100.0 µl
 TEMED 1.67 µl
 APS (100mg/ml) 50.0 µl

12. +
Materials needed for zymography
 Separating Gel 10%
 dH2O 1.788 ml
 Separating buffer 1.67 µl
 Acrylamide 2.22 µl
 20% SDS 50.0 µl
 10% Glycerol 100.0 µl
 Fibrinogen 50.0 µl
 Plasminogen 40.0 µl
 TEMED 1.67 µl
 Thrombin 2.0 µl
 Amm.Per sulfate 50.0 µl
 Stacking Gel 4%
 dH2O 2.975 ml
 Stacking buffer 1.25 ml
 Acrylamide 0.662 ml
 20% SDS 25.0 µl
 10% Glycerol 25.0 µl
 TEMED 0.25 µl
 Amm.Per sulfate(100mg/mL) 25 .0 µl

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

14. +
Procedures

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

16. +
Results
0.000
0.500
1.000
1.500
2.000
2.500
3.000
0 2 4 6 8 10 12 14 16 18
Absotbance
Volume
Chromatogram
Eluate
Wash
Load

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

18. +
Results
y = 1177.9x - 5.0193
R² = 0.9478
0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
0.000 0.050 0.100 0.150 0.200 0.250 0.300
Enzymeactivity(IU/mL)
Abs at 405 nm
Enzyme Activity
y = 1.4221x - 0.0141
R² = 0.993
-0.200
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0.000 0.200 0.400 0.600 0.800
AmountofProtein
Absorbancy at 562nm
Protein Estimation

19. +
Results
Ladder
HeLa
Load
HeLa
Spent Eluate
Plasminogen
Activator
SDS-Page with Silver
Staining (Without
Protein Estimation)
Ladder
HeLa
Load
HeLa
Spent
E
l
u
a
t
e
Plasminogen
Activator
Keratin
SDS-Page with Silver
Staining (With Protein
Estimation)

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
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an Interdigitated Microelectrode. Journal of Food Protection. [Internet];
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 Frimpong Reynolds A, Hilt J Zach. 2010.Magnetic nanoparticles in
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