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Purification of HQ-Tagged Proteins 157
2. Centrifuge the spin column with the collection tube for 5soruntil the liquid
clears the spin column.
3. To save the flow through, remove the spin column from the collection tube and
transfer the flow through from the collection tube to a new 1.5 ml microcentrifuge
tube. Otherwise, discard the flow through.
4. Place the spin column back onto the collection tube. Add 500 μl of HisLink
Binding/Wash Buffer plus the same amount NaCl used in binding to the spin
column, then cap the spin column. Centrifuge for 5soruntil the buffer clears the
spin column. Discard the flow through. Repeat for a total of two washes.
5. Take the spin column off the collection tube and wipe the base of the spin column
with a clean absorbent paper towel to remove any excess buffer.
6. Place the spin column onto a new 1.5 ml microcentrifuge tube. Add 200 μl of
HisLink Elution Buffer. Cap the spin column and tap or flick it several times
to resuspend the resin. Wait for 3 min.
7. Centrifuge the HisLink Spin Column and microcentrifuge tube at 14,000 rpm
for 1 min to collect the eluted protein.
3.3.3. Vacuum Protocol for Spin Columns
1. Place a HisLink Spin Column onto a vacuum adapter and then attach the adapter
to a vacuum port. Use a wide-bore pipette tip to transfer the lysate and resin to
the spin column. Any unused ports on the vacuum manifold must be closed for
the manifold to work properly.
2. Apply a vacuum for 5soruntil the lysate clears the spin column.
3. Add 500 μl of HisLink Binding/Wash Buffer plus the same amount of NaCl
used in binding to the spin column. Apply a vacuum for 5 s. Repeat for a total of
two washes.
4. Take the spin column off the vacuum adapter and wipe the base of the spin column
with a clean absorbent paper towel to remove any excess buffer.
5. Place the spin column onto a new 1.5 ml microcentrifuge tube. Add 200 μl of
HisLink Elution Buffer. Cap the spin column and tap or flick it several times
to resuspend the resin. Wait for 3 min.
6. Centrifuge the spin column with the 1.5 ml microcentrifuge tube at 14,000 rpm
for 1 min to collect the eluted protein.
3.4. Large-Scale Column-Based Lysis and Purification of HQ-Tagged
Proteins Using HisLink Resin
3.4.1. Lysis of Pelleted Bacterial Cells Using Sonication
1. Centrifuge bacterial culture at >10,000 × g for 15 min. Remove the supernatant
completely.
2. Resuspend pellet in cell lysis reagent or 100 mM HEPES + 10 mM imidazole, pH
7.5, at 10 to 50 fold concentration of the cell culture, depending on the amount of
protein expressed in the culture.