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