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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
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
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
Editor's Notes
The main goal for this proposal is to understand how the ligand density on an affinity membrane and its pore size can be manipulated to obtain the optimal protein binding.
This goal will be attained via the following specific aims:
1.
2.
I gave you factors that why using is better to use membrane however in industrial sectors they still using the traditional chromatography for separation, one of the major reason behind this has been a lack of availability of appropriately modified membrane.
Affinity membrane in particular are very promising alternative to affinity bead base chromatographic media.
Also the pose size and the ligand density.
This two are really important to determined the membrane performance however the exact relation between protein binding capacity and the pore size/ligand density is not clear.
Synthesis of membranes (Otaño).
2. Modification of membranes (Fasoli).
3. Application to protein binding (Bansal)
We have the membrane that have been modified
Elution buffer: 2.0 M NaCl
Regeneration buffer1: 0.1M Tris Hcl, 0.5 M NaCl, pH 8.5
Regeneration buffer2: 0.1M Sodium acetate, 0.5 M NaCl, pH 4.5