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Purification of HQ-Tagged Proteins 165
identify post-translational modifications, protein profiles, protein–protein interactions
and protein–small molecule interactions and study protein structure
and function (21–24). HQ-tagged protein purification systems provide large
amounts of protein or small amounts of multiple proteins for study. However,
the elution buffers used in these systems contain salts (e.g., imidazole) that
cannot be used in MS analysis. To be compatible with MALDI-TOF MS
analysis, eluted samples need to undergo tedious dialysis methods or size
exclusion separation techniques to remove salts. We have developed various
methods for the elution of HQ-tagged proteins. These elution conditions allow
direct MS analysis and provide clean MS data necessary for high-throughput
analysis using MALDI-TOF MS.
3.9.1. Elution from Magnetic Particles
1. After washing the MagneHis Ni Particles with MagneHis Binding/Wash
Buffer, wash the Ni Particles twice with 150 μl of 10 mM ammonium acetate (pH
7.5) or 30% ethanol.
2. Elute with 100 μl of 0.1% TFA in 50% acetonitrile.
3. Dry sample in a Speed Vac® concentrator or air-dry.
4. Resuspend the sample in the solvent or buffer that will be used for MS analysis.
3.9.2. Elution from Non-Magnetic Particles
1. After binding, wash the resin twice with 500 μl of 100 mM HEPES (pH 7.5) plus
0.5 M NaCl to decrease non-specific binding.
2. Wash the HisLink Spin Columns four times with 500 μl of double-distilled
water to remove the NaCl and buffer from the resin.
3. Elute with 200 μl of 0.1% TFA in 50% acetonitrile.
4. Dry sample in a Speed Vac® concentrator or air-dry.
5. Resuspend the sample in the solvent or buffer that will be used for MS analysis.
4. Notes
1. FastBreak Cell Lysis Reagent was designed to lyse cells without the addition
of lysozyme. Lysozyme, if added, will co-purify with the HQ-tagged protein
unless 500 mM NaCl is added to the wash buffer. These lysis methods have
been used successfully with Luria-Bertani (LB) and Terrific Broth (TB) medium.
Some bacterial strains may require a freeze-thaw cycle to achieve maximal lysis.
This can be achieved by freezing the cell pellet or culture at –20°C for 15 min
or –70°C for 5 min.
2. Some proteins purify more efficiently from a cell pellet. Test both direct lysis and
lysis of a bacterial culture to determine which is optimal for the target protein.
3. Resuspend lyophilized DNase I (cat. no. Z358B, Promega) with 80 μl doubledistilled
water.