TITLE: Microscale Thermophoresis determination of Rab29 binding to LRRK2 Armadillo Domain
AUTHORS: Edmundo G. Vides, Suzanne Pfeffer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Several labs have shown that Rab29 GTPase can recruit LRRK2 to the surface of the Golgi complex. We describe here a method to monitor direct binding of Rab29 to the LRRK2 N-terminal Armadillo domain using Microscale Thermophoresis. This method utilizes purified, His-tagged LRRK2 Armadillo residues 1-552-labeled with NanoTemper Second Gen NHS-Red 647 dye and bacterially expressed Rab29 protein.  Binding is detected using a Nanotemper Monolith NT-115 instrument.</div></div>

[STEPS]
?. [Buffer exchange Proteins]
Using either Nanotemper “A-Column” provided in labeling kit or (our preference)  Zeba Spin column 7MWCO. Twist off bottom of column and loosen cap.
0.5 mL
?. [Buffer exchange Proteins]
Prespin column: Centrifuge at  for  to remove storage solution.
4 °C
Centrifuge: 1500 34
?. [Buffer exchange Proteins]
Apply  of your labeling buffer to center of resin bed and centrifuge at  for . Do this 4X.
300 µl
Centrifuge: 1500 34
?. [Buffer exchange Proteins]
Place column in fresh collection tube. Apply  of your sample to resin and spin at  for .
100 µl
Centrifuge: 1500 34
?. [Buffer exchange Proteins]
Collect the protein flowthrough.
?. [Labeling of LRRK2 Armadillo]
Do the labeling with 2nd Generation NHS RED label from Nanotemper.
?. [Labeling of LRRK2 Armadillo]
Resuspend the dye in  DMSO as per protocol from Nanotemper to make it  final stock concentration.
25 µl
?. [Labeling of LRRK2 Armadillo]
Set up  labeling reaction using a 3:1 ratio of dye to protein; use the dye at final concentration of ; Buffer-exchanged Armadillo is used at a final concentration of  .
100 µl
Note: reducing reagent will interfere with labeling. Low concentration TCEP is acceptable
?. [Labeling of LRRK2 Armadillo]
Add above buffer to bring volume to  final volume, mix by flicking tube.
100 µl
?. [Labeling of LRRK2 Armadillo]
Incubate for  in dark at .
4 Room temperature
?. [Labeling of LRRK2 Armadillo]
Desalt excess dye using column B provided in kit or another Zeba Spin column as before.
?. [Labeling of LRRK2 Armadillo]
Spin labeled sample hard  for  at  to remove any aggregates.
Centrifuge: 14000 34
4 °C
?. [Labeling of LRRK2 Armadillo]
Take absorbance  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.
280
?. [Binding Reaction]
In , prepare  Armadillo-647 from labeled stock, BSA to final concentration of  and Tween-20 to 0.01%.
20 µl
?. [Binding Reaction]
18x  Lobind tubes are set out in tube rack and label with final concentrations of Rab29 (#1-16), Test (initial fluorescence test #17), and  Armadillo-647 (no Rab) (#18).
0.5 mL
?. [Binding Reaction]
Pipet  binding buffer into bottom of Tubes 2-17. Make sure to cap, to prevent evaporation.
5 µl
?. [Binding Reaction]
To make 2:1 dilution series; Add  of unlabeled Rab29 to Tube 1. Pipet up and down and then take  from Tube 1 and add to Tube 2. Pipet up and down slowly 5 times, being careful to hold tip at the top of volume, raising and lowering it, to avoid any of solution sticking to outside of tip (try to keep trituration number consistent with all samples). Repeat by taking  of Tube 2 and add to Tube 3. Continue until Tube 17. Use the same tip the whole way. Do best to avoid solution sticking to inner Lobind tube walls. At end, your dilution series should have  in each sample.
15 µl
10 µl
10 µl
5 µl
?. [Binding Reaction]
Add  of  Armadillo to each  sample for a final volume of . Triturate each 5-10 (again be consistent across samples; change tips for each tube.
1 µl
5 µl
6 µl
?. [Binding Reaction]
Quick spin each tube (1-16) and incubate in the dark for  at  to allow binding.
4 Room temperature
?. [Binding Reaction]
While samples are incubating, take Test (tube 17), and load into a NT.115 premium treated capillary. Set into position 16 of monolith sample tray and hit “Start Cap Scan” to measure initial fluorescence. Adjust LED power to achieve a range of 800-1200.
?. [Binding Reaction]
Set up MST project settings, usually for a new binding experiment we do 2x 20% MST, 2x 40% MST, and 2x 60% MST. 5 sec before fluorescence before MST, 30 sec MST, 2 sec fluorescence after MST, and 2 sec delay. Enter protein titration concentrations with dilution ratio.
?. [Binding Reaction]
After 30 min, hard spin tubes #1-16 at  for  at . Load into NT.115 premium treated capillaries (Nanotemper Technologies).
Centrifuge: 14000 34
4 °C
?. [Binding Reaction]
Place capillaries into machine and press “Start Cap Scan + MST measurements.
Data analysis is performed with NTAffinityAnalysis software (NanoTemper Technologies) in which the binding isotherms are derived from the raw fluorescence data and then fitted with both NanoTemper software and GraphPad Prism to determine the KDusing a non-linear regression method. The binding affinities determined by the two methods are similar.