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Mn ps final oral presentaion rise corrected

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  • 1. PROTEIN SEPARATION USING MAGNETIC NANOPARTICLES Anthony Maldonado Castro Félix Vallés 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, 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.
  • 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.  “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)
  • 9. 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)
  • 10. Fibrinolysis- breakdown of fibrin clot to prevent thrombus formation α2- antiplasmin tP A Plasminoge n Plasmin Fibrin clot degradatio n
  • 11. 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)
  • 12. 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)
  • 13. Isolate Plasminogen Activator (PA) from mammalian cell culture broth. Objectives:
  • 14. Plasminogen Activator (PA) will be isolated from mammalian cell culture broth. Hypothesis:
  • 15. Procedure Suspension Equilibration Incubation Regeneration Eluates Wash Desalting Absorbance SDS and Zymography
  • 16. Results
  • 17. 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
  • 18. Zymography Gel
  • 19. 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
  • 20. 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.
  • 21. SDS - PAGE The second SDS PAGE was stained using silver stain. This time elution 1 can be easily distinguished.
  • 22. 0 0.5 1 1.5 2 2.5 1.47 1.71 13 16.2 18 Absorbance(562nm) Sample Volume of each sample (µl)
  • 23. Conclusions  The use of magnetic nanoparticles is an efficient method for protein separation.  Have an efficient reusability.
  • 24. Acknowledgements  Dr Vibha Bansal  RISE Program  Alexandra Rosado  Osvaldo Vega  Natalia Espada  José J. Rosado  Mariana León
  • 25. PROTEIN SEPARATION USING MAGNETIC NANOPARTICLES Anthony Maldonado Castro Félix Vallés Dr. Vibha Bansal RISE Program