Bio-Rad Aurum Serum Protein Mini Kit - Manual (Page 3)

Section 1
Introduction
The Aurum serum protein mini kit contains Micro Bio-Spin
TM
columns filled with
a mixture of Affi-Gel

Blue and Affi-Gel protein A. This resin blend allows for
the simultaneous removal of both albumin and immunoglobulin (IgG) in a single
step from serum or plasma samples prior to two-dimensional (2-D)
polyacrylamide gel electrophoresis (PAGE).
Proteins in serum and other biological fluids are difficult to resolve by 2-D
PAGE, largely due to the abundance of serum albumin and IgG. In human
serum, albumin constitutes 50–70% of the total protein and IgG constitutes
10–25%. The presence of these proteins obscures other proteins in the gel
and limits the amount of proteins in the serum that can be resolved by 2-D
analysis. Furthermore, these proteins have wide pI and molecular weight
ranges, further reducing resolution and masking many proteins of potential
interest. Theoretically, removing 90% of the albumin and IgG from serum
should reduce the protein load by 70–80%, thus allowing the application of
3–4 times more serum and at the same time significantly improving the
resolution of polypeptide spots on 2-D gels.
Affi-Gel Blue affinity gel is a beaded, cross-linked agarose gel with covalently
attached Cibacron Blue F3GA dye. It has a capacity for albumin binding of
greater than 11 mg/ml. Affi-Gel Blue gel has been utilized to separate and
purify a number of different serum and plasma proteins (Gianazza and Arnaud
1982, Herman and Roberts 1980) and has been used as a first step in the
purification of serum proteins by removing the major serum constituent,
albumin (Burgett and Greenley 1977). The major advantage of Affi-Gel Blue in
the current application is its high albumin capacity. This product has been
used to rapidly remove albumin from multiple human serum samples prior to
2-D analysis (Rengarajan et al. 1996). The disadvantage of this ligand is its
possible lack of specificity, based on its numerous applications. Although the
binding of serum proteins other than albumin and IgG cannot be excluded, the
current product has been developed to minimize nonspecific adsorption.
Binding conditions have been optimized to saturate the matrix binding sites
with the very high-affinity albumin molecules.
Protein A, from Staphylococcus aureus, has the property of binding with high
specificity the Fc region of IgG molecules (Kronvall and Williams 1969). When
coupled to agarose beads, protein A has been extensively used to bind and
purify IgG species from various mammalian species (Lindmark et al. 1983).
Affi-Gel protein A has a binding capacity of greater than 15 mg human IgG/ml
gel.
1
4110021B.qxd 8/14/2003 2:50 PM Page 1