Western Blot in Mouse Brain Tissue for detecting pRab proteins

Freeze freshly dissected brain tissue on dry ice and store at -80°C

Make lysis buffer - keep on ice

A	B	C	D
Stock solutions	for 10mL	for 15mL	For 20mL
Tris HCl 1M pH7.5	500uL	0.75 mL	1.0 mL
EGTA 0.1M	100uL	0.15 mL	0.2 mL
Triton X 100	100uL	0.15 mL	0.2 mL
PIC I 100X	100uL	0.15 mL	0.2 mL
PIC III 20X	500uL	0.75 mL	1.0 mL
1.35M Sucrose (5X)	2.0mL	3.0 mL	4.0 mL
Complete EDTA-free protease inhibitor cocktail   (Sigma-Aldrich Cat #11836170001)	1 tablet	1 tablet	2 tablets
water		8.55
Total			15.0

Remove tissue from -80°C and keep on dry ice before starting experiment

Weigh each tissue sample and record mass

Transfer each samples to clean 5mL test tube for homogenization

Place samples on ice and add appropriate amounts of lysis buffer based on sample mass

For regional brain dissections add 1000µL of chilled lysis buffer for every 100mg of brain tissue

For organ tissue add 1000µL of chilled lysis buffer for every 100mg of brain tissue

For hemi brains or whole brains add 600µL of chilled lysis buffer for every 100mg of brain tissue

Allow samples to thaw in chilled lysis buffer for about 5 minutes

Check samples with a clean pipette tip. If tissue has thawed, move onto the next step.

Use a tissue homogenizer to lyse the tissue

10 seconds homogenization at maximum speed

tissue should be kept on ice during and after homogenization

Allow tissue to rest on ice for 0h 10m 0s - 0h 15m 0s after homogenization

Bubbles should disappear during this time

After 0h 10m 0s - 0h 15m 0s check lysate. If there are tissue chunks repeat homogenization step.

Transfer the samples to Eppendorf tubes and centrifuge at 4°C during 0h 30m 0s at 21.000rcf,0h 0m 0s

Remove samples from centrifuge and keep on ice

Transfer supernatant to new Eppendorf tube

Transfer 15µL of supernatant to new Eppendorf tube for Bicinchoninic acid (BCA) assay to measure protein concentration

Store supernatant at -80°C until ready to run western blot

Save the pellet and store at -80°C - pellet contains triton X insoluble proteins

Perform BCA analysis to measure protein concentrations

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 50mL:

7.75mL 2M Tris pH6.8

25mL glycerol

5gSDS

12.5mL 2-Mercaptoethanol

Fill with miliQ H₂O to 50mL (approx.4.75mL).

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.

Denature the proteins at70°C for 0h 10m 0s in lysis buffer / Lammeli sample buffer mix

Load 80µg of protein into each well of SDS page gel

For Rab proteins use 12.5% acrylamide gels

For LRRK2 use 7.5% acrylamide gels

Run gels at 120V

For Rab proteins run gels 1h 0m 0s

For LRRK2 run gels 1h 45m 0s

Transfer gels at 20V 1h 0m 0s at Room temperature

Wash membranes in H₂Or for 0h 2m 0s then 5 min in TBST

Wash membranes in TBST for 0h 5m 0s

Incubate membrane in Ponceau S solution (Sigma P7170) for 0h 5m 0s

Rinse membrane in H₂O and image

Wash 2 times in TBST for 0h 5m 0s each wash

Incubate membrane in 5% milk in TBST (BioRad 1706404XTU) for 1h 0m 0sat Room temperature

Wash 2 times in TBST for 0h 2m 0s each wash

Incubate 1h 0m 0s at 4°C with primary antibody diluted in 5% milk in TBST

A	B	C	D
Target	Species	Manufacturer	Dilution
pRab10	rabbit	Abcam ; ab230261	1:500
Total Rab10	rabbit	Cell Signaling ; 8127S	1:1000
pRab8a	rabbit	Abcam ; ab230260	1:500
pRab12	rabbit	Abcam ; ab256487	1:500
Total Rab12	rabbit	Protein Tech ; 18843-1-AP	1:1000
GAPDH	mouse	Protein Tech ; 60004-1-Ig	1:5000
Actin	mouse	Milipore; MAB1501	1:2000
LRRK2	rabbit	Abcam ; ab133474	1:1000
p935 LRRK2	rabbit	Abcam ; ab133450	1:1000
p1292 LRRK2	rabbit	Abcam ; ab203181	1:1000

Primary antibodies for Rab proteins and LRRK2

Wash 3 times in TBST for 0h 5m 0s

Dilute HRP-conjugated secondary antibodies 1:10,000 in 5% milk in TBST

A	B	C
goat anti rat	112-035-175	Jackson Immunoresearch
goat anti mouse	115-035-174	Jackson Immunoresearch
mouse anti rabbit	211-032-171	Jackson Immunoresearch
goat anti guinea pig	ab97155	Abcam

Secondary Antibodies

Wash membrane 3 times in TBST for 0h 5m 0s and once in TBS

Wash membrane in TBS for 0h 5m 0s

Image using ECL reagents (Amersham)

use 500µL of solution A and 500µL solution B for each membrane

Develop for at least 0h 1m 0s before imaging