Apr 12, 2023
Cell culture and Western blot
- Andrea Dickey1,
- rabrisch1
- 1UCSD Department of Cellular and Molecular Medicine
Protocol Citation: Andrea Dickey, rabrisch 2023. Cell culture and Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9bozg8j/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: April 11, 2023
Last Modified: April 12, 2023
Protocol Integer ID: 80356
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
Abstract
Protocol for detecting Rab7a phosphorylation at S72 by LRRK1 and LRRK1 mutants in HEK 293T cells.
Cell culture and transfection
Cell culture and transfection
1d 0h 15m
1d 0h 15m
Day - 1
Split 293T cells in 6-well dishes 24:00:00 before transfection at 200K/well in 2 mL DMEM (with FBS and with P/S)
1d
Day - 2
Transfection
Warm PEI and Opti-MEM at RT for 00:15:00
Label a tube for each well.
15m
Add DNA (500 ng of GFP-Rab7 + 1 µg of LRRK1 construct of interest) to 150 µL of Opti-MEM and gently vortex for 5 sec
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 00:15:00 .
15m
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
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
Cell lysis
Cell lysis
1d 12h
1d 12h
36:00:00 after transfection, begin cell lysis
1d 12h
Wash plate on ice with cold PBS 1x
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)
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 °C for 00:10:00 ), 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 µL 4x LDS buffer
10m
Western blot
Western blot
1h
1h
SDS-PAGE with Bis-Tris gel and MOPS running buffer
Load a 4-12% Bis-Tris gel with 25 µL of prepared lysate in sample buffer and run at 180V for ~ 00:50:00 or until dye front has reached the bottom of the gel.
50m
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 Overnight at 4 °C
Rinse 3x with TBST for 00:05:00
5m
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
Rinse 3x with TBST for 00:05:00
5m
Image on LiCor Odyssey CLx