Download - Vero feb2015
Understanding the effect of the ligand density and pore size on the protein binding capacity of affinity membranes.
Verónica Forbes Sánchez
Department of Chemistry
Dr. Vibha Bansal
Objectives1. To study the effect of ligand density
on protein binding capacity of affinity modified membranes.
2. To study the effect of pore size on protein binding capacity of affinity modified membranes.
Why we are using membrane?
▫Help to overcome the diffusion resistance▫High pressure drop▫Lower cost of production▫Reusable▫Highly accessible surface area
Affinity membraneIn the processes of making available new membrane for separation processes few things need to be considered:
Type of membraneLength of spacer armType of ligand
Pore size and Ligand densityDirectionally proportional relationship between ligand
density and the protein binding capacity??Higher ligand density more binding site available for
proteins1.Ligand density in turn depends on pore size which is also
known to affect the dynamic binding capacity of the membrane2.
Researchers concluded that for a given protein, the ligand density and the pore size need to be optimized.
1. Fogle, J.; Mohan, N.; Cheung, E.; Persson, J. Journal of Chromatography A 2012, 1225, 62.2. Francis, P.; von Lieres, E.; Haynes, C. Biotechnology and bioengineering 2012, 109, 615.
Pore size and Ligand density
Membrane large pore size
Membrane smaller pore size
Overview of project
Synthesis of membranes
Modification of membrane
Application to protein binding
Membrane design: PABA linked RC membrane
NH
Methodology: Application of affinity ligand to modified membrane to
isolation of PA.
PA Binding
Equilibrium
Washes
Elution
Regeneration
Regeneration
Methodology: Application of affinity ligand to modified membrane to
binding of PA.
Analytical methods
Analytical Methods
1Smith, 2005. Nat. Methods 2: 712Johnsen, et al., 1999. Infect. Immun. 67: 1072
Procedures ObjectiveAbsorbance at 280 nm To determine which of
the eluted fractions have protein.
Protein Estimation1
-BCA MethodTo determine the
amount of protein in sample.
Enzyme Activity2
-Chromogenic Assay
To measure the quantity of active
enzyme in a sample.
Electrophoretic Analysis
Acknowledgement• Vibha Bansal Ph.D• Ezio Fasoli Ph.D and his lab. • Rise program• NIH-INBRE• Dept. of Chemistry, UPR-Cayey
Questions
Understanding the effect of the ligand density and pore size on the protein binding capacity of affinity membranes.
Verónica Forbes Sánchez
Department of Chemistry
Dr. Vibha Bansal