Binding of Rab29 to the LRRK2 Armadillo domain by Microscale Thermophoresis
Edmundo Vides, Suzanne R Pfeffer
Abstract
Microscale thermophoresis (MST) is a powerful tool to measure the affinities of interactions between proteins. We present here our method for determining the binding of Rab29 GTPase to the LRRK2 (Leucine rich repeat kinase 2) N-terminal Armadillo domain. Work from several labs has shown that Rab29 can recruit LRRK2 to the Golgi, where it normally resides, or to other compartments, when artificially relocalized to another cellular compartment. MST has enabled us to define the precise binding site for Rab GTPases on the LRRK2 Armadillo domain.
Steps
Buffer exchange isolated Proteins
Buffer : 50mM Hepes pH 8, 100mM NaCl, 5mM MgCl2, 20µM GTP, 0.2mM TCEP, 5% glycerol
- Use either a Nanotemper “A-Column” provided in labeling kit or (our preference) 0.5 ml Zeba Spin column 7MWCO. Twist off bottom of column and loosen cap.
- Prespin column: Centrifuge at 4°C 1500xG for 1 min to remove storage solution.
- Apply 300µL of buffer to center of resin bed and centrifuge at 1500xG for 1min. Do this 4X.
- Place column in fresh collection tube. Apply 100µL of your sample to resin and spin at 1500xG for 2min.
- Protein is in collected flowthrough.
'font-size:12.0pt;line-height:107%;font-family:Arial;mso-fareast-font-family:
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font-family:Arial'>Labeling is done with 2nd Generation NHS RED
label from Nanotemper
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font-family:Arial'>Dye is resuspended in 25µl DMSO as per protocol from Nanotemper
to make it 600µM final stock concentration
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font-family:Arial'>Set up 100µL labeling reaction using a 3:1 ratio of dye to
protein; Dye is used at final concentration of 30µM; buffer exchanged Armadillo
is used at a final concentration of 10µM (note: reducing reagent will interfere
with labeling. Low concentration TCEP is acceptable)
'font-size:12.0pt;line-height:107%;font-family:Arial;mso-fareast-font-family:
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font-family:Arial'>Add above buffer to bring volume to 100µL final volume, mix
by flicking tube
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font-family:Arial'>Incubate for 30min in dark at room temperature
'font-size:12.0pt;line-height:107%;font-family:Arial;mso-fareast-font-family:
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font-family:Arial'>Desalt excess dye using column B provided in kit or another
Zeba Spin column as before
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font-family:Arial'>Spin labeled sample hard 14000xg for 10min at 4°C to remove
any aggregates
'font-size:12.0pt;line-height:107%;font-family:Arial;mso-fareast-font-family:
Arial'>
font-family:Arial'>Take absorbance 280nm of labeled protein and use extinction
coefficient and the Nanotemper Degree of Labeling (DOL) calculator to determine
concentration and DOL https://nanotempertech.com/dol-calculator/. DOL should be
between 0.5-1
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Labeling
of LRRK2 Armadillo
