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154 Godat et al.
2. NaCl (5 M).
3. Vacuum pump.
4. Vacuum holder.
2.9. Mass Spectrometry Elution Conditions from Magnetic Particles
1. Ammonium acetate, 10 mM, pH 7.5.
2. Ethanol, 30%.
3. TFA (trifluoroacetic acid), 0.1%, in 50% acetonitrile.
4. Speed Vac® Concentrator.
2.10. Mass Spectrometry Elution Conditions from Non-Magnetic
Particles
1. HEPES, 100 mM, pH 7.5 + 500 mM NaCl.
2. Double-distilled water.
3. TFA, 0.1%, in 50% acetonitrile.
4. Speed Vac® Concentrator.
2.11. Cloning Vectors
The HQ-tag containing Flexi® Vectors are available for the cloning of
desired proteins (9). The HQ tag can be appended to any protein-coding region
using Flexi® Vectors designed for bacterial or in vitro protein expression.
Flexi® Vectors are designed for rapid, high-fidelity transfer of protein-coding
regions between vectors containing various expression or peptide tag options
(9). These vectors enable expression of native or fusion proteins to facilitate
the study of protein structure and function.
3. Methods
3.1. Purification of HQ-Tagged Proteins Expressed in Bacterial Cells
3.1.1. Preparation of Bacterial Cells
Bacterial cultures can be grown in tubes, flasks or 96-well plates. Grow the
culture containing the HQ-tagged fusion proteins to an OD 600 nm between 0.4 and
0.6 and then induce protein expression. For IPTG (Isopropyl -D-thiogalactoside)
induction, add IPTG to a final concentration of 1 mM and incubate at 37ºC
for3hor25ºC overnight. Determine the OD 600 nm of the fresh bacterial culture.
3.1.2. Bacterial Cell Lysis
There are several methods for lysis of bacterial cells such as mechanical
disruption (sonication or French press), enzymatic methods (lysozyme) and