Biology Reference
In-Depth Information
debris in the supernatant that may clog up the column. Save samples of
supernatants to troubleshoot using SDS-PAGE gel.
7. Mix the supernatant of the lysed cells in batch with Ni 2 þ -NTA beads
equilibrated in lysis buffer. Agitate the beads and lysate mixture for 120 min at
4 C to bind protein to the beads.
8. Wash buffers can be made ahead of time but add the ATP right before washing
the beads. Remove unbound protein by resuspending the beads inWash Buffer-1
three times and centrifuging at 1500
g for 5 min at 4 C. Make sure the beads
settle at the bottomof the tube completely before removing the wash buffer. Save
samples of supernatants to troubleshoot using SDS-PAGE gel.
9. In between the steps to remove excess protein, prepare the spin column by
centrifuging with ddH 2 O four times.
10. Wash the beads with bound protein three times with Wash Buffer-2 by
centrifuging at 1500
g for 5 min at 4 C. Save samples of supernatants to
troubleshoot using SDS-PAGE gel.
11. After the washes are complete, pour the beads into a chromatography column.
12. To elute the protein, incubate the beads with 1 ml of elution buffer for 10 min
and then allow it to flow through the column.
13. Repeat this step three times to recover as much protein as possible.
14. To identify which fractions the protein eluted in, place 2 m l of each elution
fraction on filter paper and stain with Coomassie blue. Destain for 30 s.
15. Pool all fractions that contain the protein in a spin column to buffer exchange
into Severing Buffer I and concentrate into 250 m l final volume. See Fig. 13.2
for a schematic depiction of purification process.
13.1.1.4 Concentration determination
13.1.1.4.1 Buffers, reagents, and equipment
￿ 100 m l of Severing Buffer I
￿ Pierce BCA protein assay kit (Thermo Fisher Scientific, 23227, Agawam, MA)
￿ Microplate Reader (BioTek, Synergy 2 Multi-Mode, Winooski, VT)
￿
96-well plate with flat bottom wells (Thermo Fisher Scientific, 12565501,
Agawam, MA)
13.1.1.4.2 Detailed procedures
The concentration of GFP-katanin is determined by using the Pierce BCA protein
assay kit following the microplate protocol. Once we know the concentration of pro-
tein, the protein remains on ice and we continue with the microscope assays.
13.1.1.5 Special considerations for severing enzymes
1. Microtubule-severing enzymes are very difficult to work with, and it can be
frustrating when they are not functional every time. In our experience, we
purify functional protein one out of every three times. Previous research
reported ability to drop freeze in LN2 and store in LN2. We have been
unable to replicate this, so we use fresh protein. While this can be a challenge,
it is useful to know that you did not do anything wrong specifically. If the
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