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

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PROTEIN SEPARATION

USING MAGNETIC

NANOPARTICLES

Anthony Maldonado Castro

Félix Vallés

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, 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.

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.

“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)

Tissue Plasminogen Activator (tPA)

(cont.)

“t-PA is a poor plasminogen activator in the

absence of fibrin. However, in the presence of

fibrin, 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)

Fibrinolysis- breakdown of fibrin

clot to prevent thrombus

formation

α2-

antiplasmin

tP

A

Plasminoge

nPlasmin

Fibrin clot

degradatio

n

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)

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)

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

Results

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

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. This might be

result of low

concentration of the rest

of the samples loaded

on to the gel.

SDS - PAGE

The second

SDS PAGE

was stained

using silver

stain. This time

elution 1 can

be easily

distinguished.

0

0.5

1

1.5

2

2.5

1.47 1.71 13 16.2 18

Absorb

ance (

562 n

m)

Sample Volume of each sample (µl)

Conclusions

Plasminogen activator can be effectively

separated from mammalian cell culture.

The use of magnetic nanoparticles is an

efficient method for protein separation.

Have an efficient reusability.

Acknowledgements

Dr Vibha Bansal

RISE Program

Alexandra Rosado

Osvaldo Vega

Natalia Espada

José J. Rosado

Mariana León

PROTEIN SEPARATION

USING MAGNETIC

NANOPARTICLES

Anthony Maldonado Castro

Félix Vallés

Dr. Vibha Bansal

RISE Program