Apr 24, 2024
Western Blot in Mouse Brain Tissue for detecting pRab proteins
- 1Van Andel Research Institute
Protocol Citation: madalynn.erb Erb 2024. Western Blot in Mouse Brain Tissue for detecting pRab proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge545jg47/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 24, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 98678
Keywords: ASAPCRN, prab proteins in mouse brain tissue, detecting prab protein, detecting prab proteins protocol, prab proteins protocol, western blot in mouse brain tissue, mouse brain tissue, western blot
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000592
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Abstract
Protocol for detecting pRab proteins in mouse brain tissue
Materials
TBS (20L)
400 mL : 1M Tris pH 7.5
600 mL : 5M NaCl
19 L : H2O
TBST (20L)
400 mL : 1M Tris pH 7.5
600 mL : 5M NaCl
19 L : H2O
20 mL : Tween 20
10X Transfer Buffer
- 700 mL mH2O
- 30.4 g Tris base
- 144 g Glycine -
Fill to 1000 mL with H2O
Store at Room temperature
1X Transfer Buffer:
- 100 mL 10X Transfer Buffer
- 200 mL Methanol
- 700 mL mH2O
Store at 4'C
10X Running Buffer :
Dissolve the following components in 500 mL H2O:
- 30.0 g Tris base
- 144.0 g glycine
- 10.0 g SDS (Sodium dodecyl sulfate)
Fill to 1000 mL with H2O
The pH of the buffer should be 8.3 and no pH adjustment is required. Store the running buffer at
room temperature and dilute to 1X before use.
5X Lammeli sample buffer
10% SDS
50% glycerol
25% 2-Mercaptoethanol
0.31M tris pH6.8
0.01% bromophenol blue
Lysate preparation
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 1000 µL of chilled lysis buffer for every 100 mg of brain tissue
For organ tissue add 1000 µL of chilled lysis buffer for every 100 mg of brain tissue
For hemi brains or whole brains add 600 µL of chilled lysis buffer for every 100 mg 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 00:10:00 - 00:15:00 after homogenization
25m
Bubbles should disappear during this time
After 00:10:00 - 00:15:00 check lysate. If there are tissue chunks repeat homogenization step.
25m
Transfer the samples to Eppendorf tubes and centrifuge at 4 °C during 00:30:00 at 21.000 rcf
30m
Remove samples from centrifuge and keep on ice
Transfer supernatant to new Eppendorf tube
Transfer 15 µL 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
Western Blot
3h 55m
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 g SDS
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.
Denature the proteins at70 °C for 00:10:00 in lysis buffer / Lammeli sample buffer mix
10m
Load 80 µg 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 01:00:00
For LRRK2 run gels 01:45:00
2h 45m
Transfer gels at 20V Overnight at Room temperature
1h
Blot membranes
2h 35m
Wash membranes in H2Or for 00:02:00 then 5 min in TBST
2m
Wash membranes in TBST for 00:05:00
5m
Incubate membrane in Ponceau S solution (Sigma P7170) for 00:05:00
5m
Rinse membrane in H2O and image
Wash 2 times in TBST for 00:05:00 each wash
5m
Incubate membrane in 5% milk in TBST (BioRad 1706404XTU) for 01:00:00 at Room temperature
1h
Wash 2 times in TBST for 00:02:00 each wash
2m
Incubate Overnight at 4 °C with primary antibody diluted in 5% milk in TBST
1h
| 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 00:05:00
5m
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 00:05:00 and once in TBS
5m
Wash membrane in TBS for 00:05:00
5m
Image using ECL reagents (Amersham)
use 500 µL of solution A and 500 µL solution B for each membrane
Develop for at least 00:01:00 before imaging
1m