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