Bio-Rad ProteinChip Fractionation and Purification Products User Manual

Page 2

Q ceramic HyperD F sorbent mimics the binding and
elution characteristics of ProteinChip Q10 arrays.
ProteinChip Q spin columns can be used for small-scale
purifications of proteins discovered on array surfaces.

Storage

Store columns at 4°C.

Technical Considerations

Recommended sample binding time is 20–40 min

Each spin column containing Q ceramic HyperD F
sorbent has a minimal protein binding capacity of
7.7 mg

Column Equilibration

ProteinChip Q spin columns contain a sorbent that
requires buffer equilibration prior to use. This equilibration
buffer should be the same as the intended sample
binding buffer. To ensure that most proteins will bind to
the column, the salt concentration should be <100 mM,
pH ~9.

1. Tap the spin column lightly to settle the sorbent to

the bottom (near the tapered end) of the column.

2. Remove the caps on the top and bottom of the

spin column.

3. Set the column upright in a 1.5 ml microcentrifuge tube.

are used that take advantage of the charge differences in
species bound to the IEC support.

ProteinChip Q (quaternary amine) spin columns contain
Q ceramic HyperD F sorbent, an anion exchange
chromatography support. This material has been
specifically designed to rapidly capture and separate
proteins and peptides from crude biological mixtures
under physiological conditions of ionic strength.

Applications

Crude biological samples contain thousands of proteins
and peptides. Their complexity presents a significant
analytical challenge for researchers evaluating in vitro
and in vivo experiments from cell lysates and plasma
samples. Strategies to reduce the complexity of these
samples are essential to gaining a better understanding
of cellular mechanisms and disease states.

IEC is a powerful approach to prefractionation of
biological samples. This technology can reduce the
presence of abundant species, greatly increase the
number of less abundant species, and enrich samples
in elements of interest. ProteinChip Q spin columns are
designed to facilitate the analysis of crude biological
samples by fractionating proteins on the basis of net
surface charge. Biological samples can be further
simplified by fractionation with ProteinChip arrays.

Introduction

Ion exchange chromatography (IEC) is the most
frequently used chromatography technique for the
separation of proteins, peptides, and other charged
biomolecules. IEC achieves separation by taking
advantage of the difference in net surface charge of these
elements in a complex biological mixture.

Surface charge is determined by the presence of weak
acidic and basic groups on a protein and as such is
highly pH dependent. Typically, at pH values below a
protein’s isoelectric point (pI), the protein acquires a net
positive surface charge and will adsorb to cation
exchange media. At pH values above the protein’s pI, the
protein acquires a net negative surface charge and will
adsorb to anion exchange media. Variations in mobile
phase pH are most often used to selectively bind a
molecule of interest to an IEC support. The ideal pH value
is one that creates a large net charge difference among
the different sample components.

Desorption of bound proteins from IEC media is achieved
by either altering the pH or increasing the salt
concentration (introducing competing ions) of the mobile
phase. The higher the surface charge of the protein, the
higher the ionic strength that is needed to bring about
desorption. Variations in the mobile phase ionic strength