Affinity Chromatography - Department of Molecular and Cellular ...

ecombinant proteins in general, refer to The Recombinant Protein Handbook: Protein
Amplification and Simple Purification from Amersham Pharmacia Biotech.
HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit
Protein G, a cell surface protein from Group G streptococci, is a type III Fc-receptor.
Protein G binds through a non-immune mechanism. Like protein A, it binds specifically to
the Fc region of IgG, but it binds more strongly to several polyclonal IgGs (Table 2) and to
human IgG 3 . Under standard buffer conditions, protein G binds to all human subclasses
and all mouse IgG subclasses, including mouse IgG 1 . Protein G also binds rat IgG 2a and
IgG 2b , which either do not bind or bind weakly to protein A. Amersham Pharmacia Biotech
offers a recombinant form of protein G from which the albumin-binding region of the
native molecule has been deleted genetically, thereby avoiding undesirable reactions with
albumin. Recombinant protein G contains two Fc binding regions.
Protein G Sepharose is a better choice for general purpose capture of antibodies since it
binds a broader range of IgG from eukaryotic species and binds more classes of IgG.
Usually protein G has a greater affinity than protein A for IgG and exhibits minimal
binding to albumin, resulting in cleaner preparations and greater yields. The binding strength
of protein G for IgG depends on the source species and subclass of the immunoglobulin.
The dynamic binding capacity depends on the binding strength and also on several other
factors, such as flow rate during sample application.
Many antibodies also interact via the Fab region with a low affinity site on protein G.
Protein G does not appear to bind human myeloma IgM, IgA or IgE, although some do
bind weakly to protein A.
Leakage of ligands from an affinity medium is always a possibility, especially if harsh elution
conditions are used. The multi-point attachment of protein G to Sepharose results in very
low leakage levels over a wide range of elution conditions.
Purification options
Binding capacity Maximum Comments
operating flow
HiTrap Human IgG, > 25 mg/column 4 ml/min (1 ml column) Purification of IgG, fragments and
Protein G HP Human IgG, >125 mg/column 20 ml/min (5 ml column)
.
subclasses, including human IgG 3
Strong affinity for monoclonal
mouse IgG 1 and rat IgG.
Prepacked columns.
MAbTrap Kit Human IgG, > 25 mg/column 4 ml/min Purification of IgG, fragments and
subclasses, including human IgG 3 .
Strong affinity for monoclonal mouse
IgG 1 and rat IgG.
Complete kit contains HiTrap
Protein G HP (1 x 1 ml), accessories,
pre-made buffers for 10 purifications
and detailed experimental protocols.
Protein G Human IgG, > 20 mg/ml medium 400 cm/h* Supplied as a suspension ready
Sepharose 4 Cow IgG, 23 mg/ml medium for column packing.
Fast Flow Goat IgG, 19 mg/ml medium
Guinea pig IgG, 17 mg/ml medium
Mouse IgG, 10 mg/ml medium
Rat IgG, 7 mg/ml medium
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from
measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
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