View - ResearchGate

Purification of HQ-Tagged Proteins 163
3.8. Automated and High-Throughput Purification
of HQ-Tagged Proteins
3.8.1. Purification Using a Minirobot: Maxwell16
The Maxwell16 Purification Instrument is an automated magnetic particle
handling device. The instrument is preprogrammed with purification protocols
and can process up to 16 samples in a single run of about 40 min. In addition,
prefilled reagent cartridges contain the buffers and resin for purification for
optimal convenience.
1. Buffer configuration in cartridge, predispensed (see Table 1).
2. Optimized up to 20 OD 600 nm bacterial culture, 2×10 6 mammalian or insect cells,
1 ml culture media or 100–200 μl wheat germ reaction.
3. Add 300 μl of elution buffer to the elution tube.
4. Follow the Maxwell16 protocol for protein purification with the necessary
modifications for HQ-tagged proteins. Use the manual protocols as guide for the
addition of NaCl.
5. Increase imidazole concentration (50–100 mM imidazole) in the washes to reduce
non-specific binding in wheat germ reactions.
3.8.2. High-Throughput Purification of HQ-Tagged Proteins
Cultures with concentrations up to 8 OD 600 nm units/ml have been successfully
used with the HisLink96 Protein Purification System.
1. To 1 ml of bacterial culture, add 100 μl of the FastBreak Reagent/DNase I
solution (see Note 10).
2. Resuspend the resin and allow it to settle. Once the resin has settled, use a
wide-bore pipette tip to transfer 75 μl of the HisLink Resin from the settled
resin bed to each well of the plate.
Table 1
Maxwell ® 16 polyhistidine/HQ tagged Protein
Purification Kit Reagent Catridge Contents
Well
Buffer
1 1 ml bacterial culture, mammalian
cells, insect cells, media or wheat
germ (added by customer) 110 μl
FastBreak Cell Lysis Reagent, 10×
2 750 μl MagneHis Ni- Particles
3–6 1 ml MagneHis Binding/Wash Buffer
7 Empty