Waters Protein-Pak Epoxy-Activated Affinity Products - Manual

[ Care and Use ManUal ]
Protein-Pak Epoxy-Activated Affinity 1
Contents
I. IntroduCtIon
a. Chemistry
b. Theory of Affinity Chromatography
c. Description of the Products
II. use of epoxy-ACtIvAted AffInIty pACkIngs
a. Coupling Buffers
b. Coupling Conditions
c. Blocking Procedure
d. Washing Procedure
e. Storage
III. usIng the MICro-ColuMn
a. Preparing the Micro-Column
b. Isolation of the Selected Product
proteIn-pAk epoxy-ACtIvAted AffInIty produCts
I. IntroduCtIon
Waters Protein-Pak™ Epoxy-Activated Affinity products are silica-
based materials with nominal 40 μm particle diameter and nominal
500 Å pore size. The packing is epoxy-activated for ligand coupling.
It is capable of rapid purification of large sample volumes at high
flow rates.
a. Chemistry
Waters Protein-Pak Epoxy Activated Affinity packing is prepared
by bonding an epoxide functionality to silica. The resulting epoxy
groups covalently bond amino, sulfhydryl, and hydroxyl moieties
on small and large ligand molecules including proteins and
peptides. The order of reactivity of the ligand nucleophiles is:
RS
– > RO – > RSH > R
1
R
2
NH > ROH.
b. Theory of Affinity Chromatography
Affinity chromatography is the only chromatographic method in which
specific molecules are isolated on the basis of their bioselective
interaction with an immobilized ligand. Other chromatographic
methods rely on reversible adsorption due to physical and/or
chemical properties of the biomolecule and the support.
Epoxy-activated affinity chromatography can be performed
through the following six steps, as shown in Figure 1. Refer to
Section IV, b Operation, to perform an affinity chromatography
separation as illustrated.
Step 1: Ligand coupling or immobilization
Step 2: Loading sample mixture
Step 3: Adsorbing target material
Step 4: Washing out impurities
Step 5: Eluting target material
Step 6: Re-equilibrating the packing material