This is a presentation about isolation techniques

1. 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

2. Objectives
1. 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.

3. Why we are using membrane?
▫ Help to overcome the diffusion resistance
▫ High pressure drop
▫ Lower cost of production
▫ Reusable
▫ Highly accessible surface area

4. Affinity membrane
In the processes of making available new membrane
for separation processes few things need to be
considered:
Type of membrane
Length of spacer arm
Type of ligand

5. Pore size and Ligand density
Directionally 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.

6. Pore size and Ligand density
Membrane large pore size Membrane smaller pore size

7. Overview of project
Synthesis of membranes
Modification of membrane
Application to protein binding

8. Membrane design: PABA linked RC
membrane
NH

9. Methodology: Application of affinity
ligand to modified membrane to isolation
of PA.
PA Binding
Equilibrium
Washes

10. Elution
Regeneratio
n
Regeneratio
n
Methodology: Application of affinity
ligand to modified membrane to binding
of PA.
Analytical
methods

11. Analytical Methods
1
Smith, 2005. Nat. Methods 2: 71
2
Johnsen, et al., 1999. Infect. Immun. 67: 1072
Procedures Objective
Absorbance at 280 nm To determine which of the
eluted fractions have
protein.
Protein Estimation1
-BCA Method
To determine the amount
of protein in sample.
Enzyme Activity2
-Chromogenic Assay
To measure the quantity of
active enzyme in a sample.
Electrophoretic Analysis

12. Acknowledgement
• Vibha Bansal Ph.D
• Ezio Fasoli Ph.D and his lab.
• Rise program
• NIH-INBRE
• Dept. of Chemistry, UPR-Cayey

13. Questions

14. 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