Cell culture and Western blot

Day - 1

Split 293T cells in 6-well dishes 24h 0m 0s before transfection at 200K/well in 2mL DMEM (with FBS and with P/S)

Day - 2

Transfection

Warm PEI and Opti-MEM at RT for 0h 15m 0s

Label a tube for each well.

Add DNA ( of GFP-Rab7 500ngof GFP-Rab7 + of LRRK1 construct of interest 1µg of LRRK1 construct of interest) to 150µL of Opti-MEM and gently vortex for 5 sec

Add 3µL 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 0h 15m 0s .

In the meantime…remove media from 6-well dish containing cells and replace with fresh 1 mL DMEM (with FBS and without P/S)…put back in 37 deg incubator until ready to add transfection mixture

After 15 min incubation, add DNA/Optimem/PEI mixture dropwise 150µL onto each well

Give a bit of a light swirl before putting back in incubator

36h 0m 0s after transfection, begin cell lysis

Wash plate on ice with cold PBS 1x

Add300µL 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)

Lift with cell lifters on ice

Pipet up lysate, put in eppendorf tube, and shake 15 mins in the cold room

Spin at MAX at 4 deg for 15 mins

Remove supernatant and make sample (boil at 95°Cfor 0h 10m 0s), 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: lysate + 10x Reducing Agent + 4x LDS buffer 65µL lysate + 10µL 10x Reducing Agent + 25µL4x LDS buffer

SDS-PAGE with Bis-Tris gel and MOPS running buffer

Load a 4-12% Bis-Tris gel with 25µL of prepared lysate in sample buffer and run at 180V for ~ 0h 50m 0s or until dye front has reached the bottom of the gel.

Rinse 3x with TBST for 0h 5m 0s

Image on LiCor Odyssey CLx

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

After transfer is complete, rinse membrane with water and allow to dry between sheets of Whatman paper.

Block in 5% milk in TBS (no Tween 20)

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)

Rock at 4°C

Rinse 3x with TBST for 0h 5m 0s

Rinse 1x with 5% milk in TBST

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