Sep 12, 2024

Public workspacePurification of Human K560-GFP motor (without B+R)

 Forked from Purification of Human K560-GFP molecular motor
DOI
dx.doi.org/10.17504/protocols.io.4r3l2qr1xl1y/v1
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093;
  • 2UCSD
Katherine Surridge
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DOI: dx.doi.org/10.17504/protocols.io.4r3l2qr1xl1y/v1
Protocol Citation: Andrea M. Dickey, Katherine Surridge 2024. Purification of Human K560-GFP motor (without B+R). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qr1xl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 12, 2024
Last Modified: September 12, 2024
Protocol Integer ID: 107543
Keywords: single molecule, kinesin, purification, microtubule, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
Abstract
K560-GFP purification protocol from the Reck-Peterson Lab based on protocol from Nicholas et al. 2014. Edited for protocols.io by Andrea Dickey and Mariusz Matyszewski in 2022 and modified in 2024 for - bind and release by Kavi Rangan and Katy Surridge.
Guidelines
All protein purification steps should be performed at Temperature4 °C unless noted otherwise. From Ni-NTA step onwards, purification steps should be completed in the dark.

Materials
Materials:
cOmplete EDTA-free protease inhibitor cocktail tablets (ROCHE)
lysozyme

Buffers:
Lysis buffer (make 100 mL):
  • Concentration50 millimolar (mM) Tris pH 7.5
  • Concentration250 millimolar (mM) NaCl
  • Concentration1 millimolar (mM) MgCl2
  • Concentration20 millimolar (mM) Imidazole
  • Concentration10 millimolar (mM) BME
  • Concentration0.5 millimolar (mM) Mg-ATP
  • Concentration1 millimolar (mM) Pefabloc

Wash buffer (make 500 mL):
  • Concentration50 millimolar (mM) Tris pH 7.5
  • Concentration250 millimolar (mM) NaCl
  • Concentration1 millimolar (mM) MgCl2
  • Concentration20 millimolar (mM) imidazole
  • Concentration10 millimolar (mM) BME
  • Concentration0.5 millimolar (mM) Mg-ATP
  • Concentration1 millimolar (mM) Pefabloc

Elution buffer (make 100 mL):
  • Concentration50 millimolar (mM) Tris pH 8.0
  • Concentration250 millimolar (mM) NaCl
  • Concentration1 millimolar (mM) MgCl2
  • Concentration250 millimolar (mM) imidazole
  • Concentration10 millimolar (mM) bME
  • Concentration0.1 millimolar (mM) Mg-ATP

Storage buffer:
  • Concentration80 millimolar (mM) PIPES pH 7.0
  • Concentration2 millimolar (mM) MgCl2
  • Concentration1 millimolar (mM) EGTA
  • Concentration10 % volume sucrose
  • Concentration0.1 millimolar (mM) DTT
  • Concentration0.1 millimolar (mM) Mg-ATP





Expression
Expression
pET17b-Kif5b(1-560)-GFP-His should be transformed into BL21(DE3)RIPL cells.
Make enough LB for at least Amount2 L of culture.

Grow an overnight starter culture.
Transfer starter culture into LB. Make sure to add proper antibiotics
Allow it to grow at Shaker200 rpm, 37°C until OD600 reaches 0.6-0.8.

Chill cells and incubator to Temperature18 °C

Add Concentration0.75 millimolar (mM) IPTG and allow it to grow at Shaker200 rpm, 18°C, 16:00:00

Harvest pellets by centrifugation, wash in ice cold PBS and freeze in liquid nitrogen until use.
Purification
Purification
2h 35m
2h 35m
Resuspend Amount2 L worth of pellets in Amount40 mL Lysis buffer supplemented with 1 cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per Amount40 mL of Lysis buffer . Also add Amount1 mL ofConcentration50 mg/mL lysozyme . Vortex vigorously to resuspend.
Incubate TemperatureOn ice for Duration00:30:00 .

30m
Lyse the resuspension by sonication on ice with pulse cyclesDuration00:00:05 on and Duration00:00:20 rest.

25s
Clarify by centrifugation Centrifigation92600 x g, 4°C, 00:30:00 in Type 70 Ti rotor (Beckman).

30m
During centrifugation, wash each Amount4 mL Ni-NTA agarose slurry with Amount20 mL Wash buffer.

Incubate the supernatant with Ni-NTA agarose beads washed with the Wash buffer. Nutate for Duration01:00:00 at Temperature4 °C in the dark (from this step onwards, keep in dark as much as possible).

1h
Apply to gravity column and wash 4 times with Amount10 mL Wash buffer each time.

Resuspend the beads in Amount1 mL elution buffer, incubate for Duration00:02:00 and elute in Amount0.5 mL increments. Repeat elution step up to 5 times (following green GFP signal)

2m
Combine peak fractions and buffer exchange on PD-10 desalting column equilibrated with Storage buffer (BRB80).
Peak fractions of the motor solution were flash-frozen at Temperature-80 °C until further use. Bind and release protocol could be used at this stage, see: dx.doi.org/10.17504/protocols.io.bp2l61xrdvqe/v1