TITLE: Cell culture and Western blot
AUTHORS: Andrea Dickey, rabrisch

[DESCRIPTION]
Protocol for detecting Rab7a phosphorylation at S72 by LRRK1 and LRRK1 mutants  in HEK 293T cells. 

[STEPS]
SECTION: Cell culture and transfection
1. Day - 1 

Split 293T cells in 6-well dishes 1440 min before transfection at 200K/well in 2 mL DMEM (with FBS and with P/S)

SECTION: Cell culture and transfection
2. Day - 2 

Transfection
SECTION: Cell culture and transfection
2.1. Warm PEI and Opti-MEM at RT for 15 min 
Label a tube for each well.
SECTION: Cell culture and transfection
2.2. Add DNA (500 ngof GFP-Rab7 + 1 µg of LRRK1 construct of interest) to 150 µL of Opti-MEM and gently vortex for 5 sec

SECTION: Cell culture and transfection
2.3. Add 3 µL room-temperature PEI to each tube containing Opti-MEM/DNA mix. Mix gently by pipetting and incubate DNA/DMEM/PEI mixture at room temperature in the hood for 15 min.

SECTION: Cell culture and transfection
2.4. In the meantime…remove media from 6-well dish containing cells and replace with fresh 1 mL DMEM (with FBS and withoutP/S)…put back in 37 deg incubator until ready to add transfection mixture
SECTION: Cell culture and transfection
2.5. After 15 min incubation, add DNA/Optimem/PEI mixture dropwise 150 µL onto each well
Give a bit of a light swirl before putting back in incubator
SECTION: Cell lysis
3. 2160 min after transfection, begin cell lysis

SECTION: Cell lysis
3.1. Wash plate on ice with cold PBS 1x
SECTION: Cell lysis
3.2. Add300 µL RIPA buffer (0.5% Triton, 50 mM Tris pH7.5, 150 mM NaCl, 0.1%SDS) with protease and phosphatase inhibitors (cOmplete mini EDTA free + PhosSTOP tablets)

SECTION: Cell lysis
3.3. Lift with cell lifters on ice
SECTION: Cell lysis
3.4. Pipet up lysate, put in eppendorf tube, and shake 15 mins in the cold room
SECTION: Cell lysis
3.5. Spin at MAX at 4 deg for 15 mins
SECTION: Cell lysis
3.6. Remove supernatant and make sample (boil at 95 °Cfor 10 min), store in -80 °C . I like to store the lysate and take some out to make a sample –I use the 4x NuPage LDS sample buffer: 65 µL lysate + 10 µL 10x Reducing Agent + 25 µL4x LDS buffer

SECTION: Western blot
4. SDS-PAGE with Bis-Tris gel and MOPS running buffer
SECTION: Western blot
4.1. Load a 4-12% Bis-Tris gel with 25 µL of prepared lysate in sample buffer and run at 180V for ~ 50 min or until dye front has reached the bottom of the gel.

SECTION: Western blot
4.2. Assemble gel with Immobilon-FL PVDF membrane for transfer according to instructions from your western blot transfer apparatus. When using fluorescence detection for Western blot it is important to use low fluorescence background membrane (Immobilon-FL or equivalent).

We activate membrane with MeOH and rinse with water.
Transfer in Western transfer buffer with Tris/Glycine and 20% MeOH at 200 mA for 4 hr at 4 °C 
SECTION: Western blot
4.3. After transfer is complete, rinse membrane with water and allow to dry between sheets of Whatman paper.
SECTION: Western blot
4.4. Block in 5% milk in TBS (no Tween 20)
SECTION: Western blot
4.5. Dilute primary antibody in 5% milk with TBST (with Tween 20)
-rabbit anti Rab7 phospho-S72 (MJF-38) at 1:1000
-mouse anti-GFP at 1:2500 (Santa Cruz) (for total Rab quantification) 
-rabbit anti-LRRK1 (ab228666) at 1:500 
-rabbit anti-GAPDH at 1:3000 (Cell signaling technologies) 
SECTION: Western blot
4.6. Rock   at 4 °C 

SECTION: Western blot
4.7. Rinse 3x with TBST for 5 min 
SECTION: Western blot
4.8. Rinse 1x with 5% milk in TBST
SECTION: Western blot
4.9. Add secondary antibodies in 5% milk with TBST and incubate at Room temperature for 1 hr 
-LiCor mouse and rabbit secondary IRdye antibodies at 1:5000
SECTION: Western blot
4.10. Rinse 3x with TBST for 5 min 
SECTION: Western blot
4.11. Image on LiCor Odyssey CLx