This document discusses separating proteins using magnetic nanoparticles. It aims to isolate plasminogen activator (PA) from mammalian cell culture broth. SDS PAGE was used to estimate protein concentration, while fibrin zymography detected enzyme activity. Preliminary results showed SDS PAGE detected protein in the load samples but not eluates. Zymography found enzyme activity in the first eluate only. Future work includes repeating the analyses with increased initial PA concentration and silver staining. Magnetic nanoparticles provide a fast, scalable, and automated method for protein separation compared to traditional techniques.

1. PROTEIN SEPARATION USING
MAGNETIC NANOPARTICLES
Anthony Maldonado Castro
Félix Valles
Dr. Vibha Bansal
RISE Program

2. 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.

3. What did we use?
SDS PAGE
 Fibrin Zymography

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.

9. Fibrinolysis- breakdown of fibrin clot to
prevent thrombus formation
PA
I
tPA
Plasminogen Plasmin
Fibrin clot
degradation

10. Isolate Plasminogen Activator (PA) from
mammalian cell culture broth.
Objectives:

11. Plasminogen Activator (PA) will be isolated from
mammalian cell culture broth.
Hypothesis:

12. Procedure
Suspension Equilibration Incubation
Regeneration Eluates Wash
Desalting Absorbance SDS and
Zymography

13. Preliminary Results

14. SDS PAGE – Protein estimation
 SDS PAGE: Protein estimation
Sample ABS 562nm Abs - Blank Concentration Conc. Average
Load HeLa
2.31 2.223 4.98654105 4.384253
Load HeLad
1.773 1.686 3.781965007
Spent HeLa
1.946 1.859 4.170031404 3.835801
Spent HeLad
1.648 1.561 3.501570211
Eluate HeLa1
0.109 0.022 0.049349484 0.026918
Eluate HeLa1d
0.089 0.002 0.004486317
Eluate HeLa2
0.095 0.008 0.017945267 0.020188
Eluate HeLa2d
0.097 0.01 0.022431584
Eluate HeLa3
0.095 0.008 0.017945267 0.01794
Eluate HeLa3d
0.673 0.586 1.314490803

15. SDS PAGE
Only the marker and the
HeLa Load can be
appreciated in the gel
image

16. 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

17. Zymography Gel

18. Future Work
 Another SDS and (possibily) a Zymography
 Increase initial TPA concentration
 Use silver staining instead of Coomassi Blue

19. Acknowledgements
 Dr Vibha Bansal
 RISE Program
 Alexandra Rosado
 Osvaldo Vega

20. PROTEIN SEPARATION USING
MAGNETIC NANOPARTICLES
Anthony Maldonado Castro
Félix Valles
Dr. Vibha Bansal
RISE Program