Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 9)

Table 3 provides a list of denaturing IMAC buffer compositions. All buffers
are 1x working buffer and placed on positions 1–7. The lysis buffer is used
for sample preparations (see Section 6), and is not used in any of the
instrument ports.
The IMAC cartridges are identical to the cartridges described in
Section 2.1, and all specifications apply to denaturing IMAC purifications.
Table 3. Formulations for buffers and solutions for denaturing IMAC
purification.
Profinia Supplied Other Working 1x Formulation, Final
Solution As Components for Denaturing IMAC Volume aAfter Position #
Needed Purification Urea Addition*
Native IMAC 2x Add 90 g urea and 6 M urea,
lysis buffer H
2
O to 250 ml 300 mM KCl, 50 mM KH
2
PO
4
5 mM imidazole, pH 8.0 250 ml N/A
Native IMAC 2x Add 90 g urea and 6 M urea,
wash buffer 1 H
2
O to 250 ml 300 mM KCl, 50 mM KH
2
PO
4
5 mM imidazole, pH 8.0 250 ml 1
Native IMAC 2x Add 72 g urea and 6 M urea,
wash buffer 2 H
2
O to 200 ml 300 mM KCl, 50 mM KH
2
PO
4
10 mM imidazole, pH 8.0 200 ml 2
Native IMAC 2x Add 72 g urea and 6 M urea,
elution buffer H
2
O to 200 ml 300 mM KCl, 50 mM KH
2
PO
4
250 mM imidazole, pH 8.0 200 ml 3
Cleaning 2x 500 mM NaCl,
solution 1 50 mM Tris, pH 8.0 125 ml 5
Cleaning 4x 500 mM NaCl,
solution 2 100 mM NaOAc, pH 4.5 125 ml 6
Storage 2x 2% C
6
H
5
CH
2
OH
solution (benzyl alcohol) 200 ml 7
*For bigger bottle to fit the buffer volume, order catalog #620-0231.
3.2 Instructions for Use
After urea addition, the solutions containing completely dissolved urea
should be filtered through 0.2 µm filters to remove particulates. The position
labeling of the bottles and insertion into the instrument are identical to that
described in Section 2.2. Note: Denaturing IMAC methods do not
use desalting buffers (or cartridges) and position 4 is left empty
or filled with water
. Refer to Figure 3 for proper positioning of the
denaturing kit bottles. In this figure, the bottles are installed and the
instrument lid is in the closed position. If less than 10 ml is needed for a
purification, the denaturing buffers containing urea can be frozen and used
within 3 months. The stored buffers should only be thawed once and
discarded after use.
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