Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 126)

Native IMAC buffers* are used for proteins that partition into the soluble fraction of bacterial
lysates and that have accessible His sequence tags. Table 10.2 provides a list of buffer
compositions. The native lysis buffer is used for sample preparation (see Appendix E), and
is not used in any of the instrument ports.
The IMAC cartridges are provided in 20% ethanol, and can be stored between 4 and 22°C
prior to use. The cartridges are packed with 1 ml of Bio-Rad Profinity
™ IMAC resin, and
typically yield 10 mg of purified protein per run (dependent upon protein expression level
and culture volume loaded). To minimize any possibility of cross-contamination, it is
suggested that individual cartridges be dedicated to the purification of a unique His-tagged
protein. The desalting cartridges are provided in water with 0.05% azide as a preservative
and can be stored between 4 and 22°C prior to use.
* IMAC buffers made with potassium salts are more stable than sodium salt-based buffers, and do not form
precipitates with long-term storage. However, to prevent the formation and precipitation of potassium-SDS complexes
in Laemmli buffer, native IMAC samples and fractions (lysate load, flowthrough, wash 1, and wash 2 samples)
should be diluted at least 1:7 in Laemmli buffer prior to electrophoresis. The desalted and purified fraction is
collected in a PBS-based buffer and only needs dilution 1:2 prior to electrophoresis.
Table 10.2. Formulations for buffers and solutions provided in the Profinia native IMAC kits.
Profinia Supplied Concentrated Working 1x Vol, Buffer
Solution Formulation Formulation ml Position
Native 2x 600 mM KCl, 100 mM 300 mM KCl, 50 mM 125 N/A
IMAC lysis KH
2
PO
4
, 10 mM KH
2
PO
4
, 5 mM
buffer imidazole, pH 8.0 imidazole, pH 8.0
Native 2x 600 mM KCl, 100 mM 300 mM KCl, 50 mM 125 1
IMAC KH
2
PO
4
, 10 mM KH
2
PO
4
, 5 mM
wash buffer imidazole, pH 8.0 imidazole, pH 8.0
1
Native 2x 600 mM KCl, 100 mM 300 mM KCl, 50 mM 100 2
IMAC KH
2
PO
4
, 20 mM KH
2
PO
4
, 10 mM
wash buffer imidazole, pH 8.0 imidazole, pH 8.0
2
Native 2x 600 mM KCl, 100 mM 300 mM KCl, 50 mM 100 3
IMAC KH
2
PO
4
, 500 mM KH
2
PO
4
, 250 mM
elution imidazole, pH 8.0 imidazole, pH 8.0
buffer
Desalting 5x 685 mM NaCl, 13.5 mM 137 mM NaCl, 2.7 mM 200 4
buffer KCl, 21.5 mM Na
2
HP0
4
, KCl, 4.3 mM Na
2
HP0
4
,
40.5 mM KH
2
P0
4
, pH 8.1 mM KH
2
P0
4
, pH 7.4
7.0 (pH 7.4 upon dilution)
Cleaning 2x 1,000 mM NaCl, 100 mM 500 mM NaCl, 50 mM 125 5
solution 1 Tris, pH 8.0 Tris, pH 8.0
Cleaning 4x 2,000 mM NaCl, 400 mM 500 mM NaCl, 100 mM 125 6
solution 2 NaOAc, pH 4.5 NaOAc, pH 4.5
Storage 2x 4% C
6
H
5
CH
2
OH (benzyl 2% C
6
H
5
CH
2
OH (benzyl 200 7
solution alcohol) alcohol)
119