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164 Godat et al.
3. Adding 200 mM NaCl prior to the addition of the HisLink Resin may reduce
non-specific binding and improve binding of HQ-tagged proteins. If NaCl is used
in binding also, use NaCl in the washes.
4. Incubate the sample and resin for 30 min, mixing frequently by vortexing or
pipetting to optimize binding.
5. Place a filtration plate onto the vacuum manifold base (see Note 11).
6. Use a wide-bore pipette to transfer the lysed lysate and resin to the filtration
plate.
7. Cover unused filtration plate wells with an adhesive plate sealer.
8. Apply vacuum to the samples for 10 s.
9. If you collected the flow through, remove the filtration plate from the manifold
collar and place the filtration plate onto the vacuum manifold base.
10. Add 250 μl of the HisLink Binding/Wash Buffer plus the same amount of
NaCl used in binding to the wells of the filtration plate. Apply vacuum for 10 s.
11. Repeat step 6 three more times for a total of four washes.
12. Place the filtration plate onto a clean absorbent towel to remove any excess wash
buffer from the ports located on the bottom of the plates.
13. Place the collection plate onto the manifold bed.
14. Place the manifold collar on the collection plate, fitting it into the pins of the
manifold bed.
15. Place the filtration plate onto the manifold collar. To prevent uneven flow or
spattering, remove the vacuum hose from the port on the manifold collar. Reattach
the vacuum hose at step 17.
16. Add 200 μl of the elution buffer. Wait for 3 min.
17. Reattach the vacuum hose to the manifold collar.
18. Collect the eluate by applying a vacuum for 1 min.
3.8.3. High-Throughput Purification Using Robotics
Both the MagneHis Protein Purification System and the HisLink96
Protein Purification System are amendable for high-throughput robotics (18).
The manual protocols can be used as a guide to develop protocols for
automated workstations and may need optimization depending on the instrument
used. Automated methods have been developed to purify proteins on several
workstations such as Beckman and Tecan and are easily scalable to accommodate
a variety of sample volumes. These protocols can be downloaded from
http://www.promega.com/automethods.
3.9. Mass Spectrometry Analysis of HQ-Tagged Proteins
Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and
other alternative methods of mass spectrometry (MS) analysis have become
essential methods of protein analysis (19,20). Using MS methods, we could