TITLE: Cell lysis and immunoblotting for protein and phospho-protein quantification
AUTHORS: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]

Here, we describe the procedure by which human iPSC-derived neurons or mouse embryonic fibroblasts (MEFs) were lysed and probed for levels of proteins of interest using Western blot.

[STEPS]
SECTION: Preparation of cell lysates
1. Quickly wash cells twice with ice-cold PBS. After the second wash, tilt the dish and completely aspirate all residual PBS. 
SECTION: Preparation of cell lysates
2. Immediately add ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer. Place cells on ice.

 

 

SECTION: Preparation of cell lysates
3. Scrape cells off the dish using a cell lifter. 

SECTION: Preparation of cell lysates
4. Centrifuge for 10 min at 17000 x g and 4 °C.

SECTION: Preparation of cell lysates
5. Add 100 µL  ß-mercaptoethanol to900 µL of 4x Protein Loading Buffer and mix well. 

SECTION: SDS-polyacrylamide gel electrophoresis
6. Load samples onto 8% (for LRRK2 protein) to 15% (for PPM1H and Rab proteins) acrylamide gels alongside Chameleon Duo pre-stained protein ladder (LI-COR). 
 

SECTION: SDS-polyacrylamide gel electrophoresis
7. Start electrophoresis at 80 V for 20 min, then increase to 120 V and electrophorese until orange dye runs out. 

SECTION: Protein transfer
8. Activate Immobilon-FL PVDF membrane by submerging in methanol for 30 s- 1 min. 

SECTION: Protein transfer
9. Fill transfer tank with ice-cold transfer buffer.
SECTION: Total protein stain, membrane blocking, and antibody incubation
10. After wet-tank transfer, let membrane dry completely for at least 60 min at Room temperature. 

SECTION: Total protein stain, membrane blocking, and antibody incubation
11. Rehydrate membrane for 1 min  in 100% methanol, then wash 5 min  in 1x TBS.

SECTION: Total protein stain, membrane blocking, and antibody incubation
12. Incubate for 5 min in Revert total protein stain (LI-COR) while gently shaking at Room temperature.

SECTION: Total protein stain, membrane blocking, and antibody incubation
13. Remove Revert total protein stain by incubating membrane in Revert reversal solution for 10 min at Room temperature  while gently shaking.

SECTION: Total protein stain, membrane blocking, and antibody incubation
14. Block membrane for 5 min  in Everyblot Blocking Buffer (Bio-Rad) at  Room temperature.

SECTION: Total protein stain, membrane blocking, and antibody incubation
15. Dilute primary antibodies in Everyblot Blocking Buffer and incubate at 4 °C 5 min. 

SECTION: Total protein stain, membrane blocking, and antibody incubation
16. Wash membrane in 1x TBS + 0.1% Tween-20 (TBS-T) at Room temperature (4 washes, 5 min each). 

SECTION: Total protein stain, membrane blocking, and antibody incubation
17. Dilute secondary antibodies 1:20,000 in Everyblot Blocking Buffer and 0.02% SDS. 
 

SECTION: Total protein stain, membrane blocking, and antibody incubation
18. Wash membrane in TBS-T at  Room temperature (4 washes, 5 min each). 

SECTION: Total protein stain, membrane blocking, and antibody incubation
19. Rinse membrane with TBS (no detergent), then image on ODYSSEY CLx imaging system. Quantify signal intensity using Image Studio Software. 
SECTION: Preparation of cell lysates
3.1. 
Transfer the cell lysate to an Eppendorf tube on ice.

SECTION: Preparation of cell lysates
3.2.  
Leave lysates on ice for 20 min to allow for efficient lysis.
 



SECTION: Preparation of cell lysates
4.1. 
Discard pellet and use clarified supernatant to determine protein concentration by BCA assay following the manufacturer’s instructions, performing all measurements in triplicates. 
SECTION: Preparation of cell lysates
5.1. 
Add complete 4x Protein Loading Buffer to cell lysates, mix well, and boil for 5 min at 95 °C. 
 



SECTION: Protein transfer
8.1.  Wash in ddH2O and equilibrate in transfer buffer. 
SECTION: Protein transfer
8.2.  
Soak sponges in methanol, wash in ddH2O and equilibrate in transfer buffer.
SECTION: Protein transfer
8.3. 
Equilibrate filter paper in transfer buffer.
SECTION: Protein transfer
8.4.  
Assemble blotting sandwich.
SECTION: Protein transfer
8.5. 
Carefully remove any air bubbles between layers using a roller. 
SECTION: Protein transfer
9.1.  Place transfer system on ice. 

SECTION: Protein transfer
9.2. 
Transfer proteins from gel onto PVDV membrane at 100 V for 75 min. 

SECTION: Total protein stain, membrane blocking, and antibody incubation
12.1. 
Wash twice with Revert wash solution, then rinse in ddH2O and image membrane on ODYSSEY CLx imaging system. 
SECTION: Total protein stain, membrane blocking, and antibody incubation
13.1.  
Rinse membrane with ddH2O.