Bio-Rad Profinia Protein Purification System Kits - Manual (Page 7)

Table 2. Formulations for buffers and solutions provided in the
native IMAC starter kit.
Profinia Solution Supplied Concentrated Formulation Working 1x Formulation Volume Position #
As
Native IMAC 2x 600 mM KCl, 100 mM KH
2
PO
4
, 300 mM KCl, 50 mM KH
2
PO
4
,
lysis buffer 10 mM imidazole, pH 8.0 5 mM imidazole, pH 8.0 125 ml N/A
Native IMAC 2x 600 mM KCl, 100 mM KH
2
PO
4
, 300 mM KCl, 50 mM KH
2
PO
4
,
wash buffer 1 10 mM imidazole, pH 8.0 5 mM imidazole, pH 8.0 125 ml 1
Native IMAC 2x 600 mM KCl, 100 mM KH
2
PO
4
, 300 mM KCl, 50 mM KH
2
PO
4
,
wash buffer 2 20 mM imidazole, pH 8.0 10 mM imidazole, pH 8.0 100 ml 2
Native IMAC 2x 600 mM KCl, 100 mM KH
2
PO
4
, 300 mM KCl, 50 mM KH
2
PO
4
,
elution buffer 500 mM imidazole, pH 8.0 250 mM imidazole, pH 8.0 100 ml 3
Desalting buffer 5x 685 mM NaCl, 13.5 mM KCl, 137 mM NaCl, 2.7 mM KCl,
21.5 mM Na
2
HP0
4
, 4.3 mM Na
2
HP0
4
,
40.5 mM KH
2
HP0
4
, 8.1 mM KH
2
P0
4
, pH 7.4 200 ml 4
pH 7.0 (pH 7.4 upon dilution)
Cleaning solution 1 2x 1,000 mM NaCl, 500 mM NaCl,
100 mM Tris, pH 8.0 50 mM Tris, pH 8.0 125 ml 5
Cleaning solution 2 4x 2,000 mM NaCl, 500 mM NaCl,
400 mM NaOAc, pH 4.5 100 mM NaOAc, pH 4.5 125 ml 6
Storage solution 2x 4% C
6
H
5
CH
2
OH 2% C
6
H
5
CH
2
OH
(benzyl alcohol) (benzyl alcohol) 200 ml 7
2.2 GST Component Descriptions
The lysis and wash buffers contained in the GST kit are formulated
from sodium salts, phosphate buffers, and EDTA, and provide optimized
binding, washing, and eluting of GST-tagged proteins. EDTA is included
as a chelating compound that protects against metalloproteases, which can
be present in
E. coli lysates. GST fusion proteins must be in a native
conformation prior to purification. The buffers used in this system are
designed for the purification of proteins that are soluble in
E. coli lysates
and that have accessible, native GST sequence tags. Table 3
provides a list of buffer compositions. The lysis buffer is used for preparing
sample preparations; it is not used in any of the instrument ports. This
buffer can be used for subsequent purifications of target protein outside of
the starter kit applications.
The GST cartridges are shipped in 20% ethanol and are packed with
1 ml Profinity GST resin. The desalting cartridges are provided in 20 mM
Bis-Tris, pH 6.5 with 0.05% azide as a preservative. The vial of lyophilized
lysate is described in sections 1.2 and 2.1, and when resuspended in 12 ml
1x GST lysis buffer, provides adequate sample for two independent
purification runs.
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