TITLE: Western Blot in Mouse Brain Tissue for detecting pRab proteins
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
Protocol for detecting pRab proteins in mouse brain tissue

[STEPS]
SECTION: Lysate preparation
1. Freeze freshly dissected brain tissue on dry ice and store at -80 °C 

SECTION: Lysate preparation
3. Remove tissue from -80 °C and keep on dry ice before starting experiment

SECTION: Lysate preparation
4. Weigh each tissue sample and record mass
SECTION: Lysate preparation
6.1. For regional brain dissections add 1000 µL of chilled lysis buffer for every 100 mg of brain tissue

SECTION: Lysate preparation
6.2. For organ tissue add 1000 µL of chilled lysis buffer for every 100 mg of brain tissue

SECTION: Lysate preparation
6.3. For hemi brains or whole brains add 600 µL of chilled lysis buffer for every 100 mg of brain tissue
SECTION: Lysate preparation
2. Make lysis buffer - keep on ice
 
 
 
SECTION: Lysate preparation
7. Allow samples to thaw in chilled lysis buffer for about 5 minutes
SECTION: Lysate preparation
7.1. Check samples with a clean pipette tip. If tissue has thawed, move onto the next step.
SECTION: Lysate preparation
6. Place samples on ice and add appropriate amounts of lysis buffer based on sample mass
SECTION: Lysate preparation
8. Use a tissue homogenizer to lyse the tissue
SECTION: Lysate preparation
8.1. 10 seconds homogenization at maximum speed
SECTION: Lysate preparation
8.2. tissue should be kept on ice during and after homogenization
SECTION: Lysate preparation
9. Allow tissue to rest on ice for  10 min - 15 min after homogenization

SECTION: Lysate preparation
9.1. Bubbles should disappear during this time
SECTION: Lysate preparation
9.2. After 10 min - 15 min check lysate. If there are tissue chunks repeat homogenization step.

SECTION: Lysate preparation
10. Transfer the samples to Eppendorf tubes and centrifuge at 4 °C during 30 min at 21.000 rcf

SECTION: Lysate preparation
5. Transfer each samples to clean 5mL test tube for homogenization
11. Remove samples from centrifuge and keep on ice
12. Transfer supernatant to new Eppendorf tube
12.1. Transfer 15 µL of supernatant to new Eppendorf tube for Bicinchoninic acid (BCA) assay to measure protein concentration

12.3. Save the pellet and store at -80 °C - pellet contains triton X insoluble proteins
12.2. Store supernatant at -80 °C until ready to run western blot

13. Perform BCA analysis to measure protein concentrations
SECTION: Western Blot
14. Dilute tissue lysates 1:5 in 5X Lammeli sample buffer

10% SDS
50% glycerol
25% 2-Mercaptoethanol
0.31M tris pH6.8
0.01% bromophenol blue

For 50 mL:
7.75 mL 2M Tris pH6.8
25 mL glycerol
5 gSDS
12.5 mL 2-Mercaptoethanol
Fill with miliQ H2O to 50 mL (approx.4.75 mL).
Add bromophenol blue

First mix Tris with 4ml H2O and add SDS.  Let it mix for about an hour.  Then add BME and glycerol and continue to mix for approximately one more hour (SDS will eventually go into solution).  QS to 50ml with additional H2O.  Add bromophenol blue, aliquot and store at -20C.
SECTION: Western Blot
15. Denature the proteins at70 °C for 10 min in lysis buffer / Lammeli sample buffer mix

SECTION: Western Blot
16. Load 80 µg of protein into each well of SDS page gel

SECTION: Western Blot
16.1. For Rab proteins use 12.5% acrylamide gels
For LRRK2 use 7.5% acrylamide gels
SECTION: Western Blot
17. Run gels at 120V
SECTION: Western Blot
17.1. For Rab proteins run gels 60 min
For LRRK2 run gels 105 min

SECTION: Western Blot
18. Transfer gels at 20V 60 min at Room temperature

SECTION: Blot membranes
19. Wash membranes in H2Or for 2 min then 5 min in TBST

SECTION: Blot membranes
20. Wash membranes in TBST for 5 min

SECTION: Blot membranes
21. Incubate membrane in Ponceau S solution (Sigma P7170) for 5 min

SECTION: Blot membranes
22. Rinse membrane in H2O and image
SECTION: Blot membranes
23. Wash 2 times in TBST for 5 min each wash

SECTION: Blot membranes
24. Incubate membrane in 5% milk in TBST (BioRad 1706404XTU) for 60 minat Room temperature

SECTION: Blot membranes
25. Wash 2 times in TBST for 2 min each wash
SECTION: Blot membranes
26. Incubate 60 min at 4 °C with primary antibody diluted in  5% milk in TBST

SECTION: Blot membranes
26.1.  
 
 
SECTION: Blot membranes
27. Wash 3 times in TBST for 5 min

SECTION: Blot membranes
28. Dilute HRP-conjugated secondary antibodies 1:10,000 in 5% milk in TBST

SECTION: Blot membranes
28.1.  
 
 
 
 
SECTION: Blot membranes
29. Wash membrane 3 times in TBST for 5 min and once in TBS

SECTION: Blot membranes
30. Wash membrane in TBS for 5 min

SECTION: Blot membranes
31. Image using ECL reagents (Amersham)
SECTION: Blot membranes
31.1.  use 500 µL of solution A and 500 µL solution B for each membrane

SECTION: Blot membranes
31.2. Develop for at least 1 min before imaging