Apr 23, 2024

Public workspaceWestern Blot in Mouse Brain Tissue for detecting pRab proteins

DOI
dx.doi.org/10.17504/protocols.io.261ge545jg47/v1
  • 1Van Andel Research Institute
madalynn.erb Erb
Open access
DOI: dx.doi.org/10.17504/protocols.io.261ge545jg47/v1
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 23, 2024
Last Modified: April 23, 2024
Protocol Integer ID: 98678
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000592
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Abstract
Protocol for detecting pRab proteins in mouse brain tissue
Materials
TBS (20L)
Amount400 mL : 1M Tris pH 7.5
Amount600 mL : 5M NaCl
Amount19 L : H2O


TBST (20L)
Amount400 mL : 1M Tris pH 7.5
Amount600 mL : 5M NaCl
Amount19 L : H2O
Amount20 mL : Tween 20


10X Transfer Buffer
  • Amount700 mL mH2O
  • Amount30.4 g Tris base
  • Amount144 g Glycine -
Fill to Amount1000 mL with H2O
Store at TemperatureRoom temperature
1X Transfer Buffer:
  • Amount100 mL 10X Transfer Buffer
  • Amount200 mL Methanol
  • Amount700 mL mH2O
Store at 4'C


10X Running Buffer :
Dissolve the following components in 500 mL H2O:
  • Amount30.0 g Tris base
  • Amount144.0 g glycine
  • Amount10.0 g SDS (Sodium dodecyl sulfate)
Fill to Amount1000 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
Lysate preparation
Freeze freshly dissected brain tissue on dry ice and store at Temperature-80 °C

Make lysis buffer - keep on ice
ABCD
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 Temperature-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 Amount1000 µL of chilled lysis buffer for every Amount100 mg of brain tissue

For organ tissue add Amount1000 µL of chilled lysis buffer for every Amount100 mg of brain tissue

For hemi brains or whole brains add Amount600 µL of chilled lysis buffer for every Amount100 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 Duration00:10:00 - Duration00:15:00 after homogenization

25m
Bubbles should disappear during this time
After Duration00:10:00 - Duration00:15:00 check lysate. If there are tissue chunks repeat homogenization step.

25m
Transfer the samples to Eppendorf tubes and centrifuge at Temperature4 °C during Duration00:30:00 at Centrifigation21.000 rcf

30m
Remove samples from centrifuge and keep on ice
Transfer supernatant to new Eppendorf tube
Transfer Amount15 µL of supernatant to new Eppendorf tube for Bicinchoninic acid (BCA) assay to measure protein concentration

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

Save the pellet and store at Temperature-80 °C - pellet contains triton X insoluble proteins
Perform BCA analysis to measure protein concentrations
Western Blot
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 Amount50 mL :
Amount7.75 mL 2M Tris pH6.8
Amount25 mL glycerol
Amount5 g SDS
Amount12.5 mL 2-Mercaptoethanol
Fill with miliQ H2O to Amount50 mL (approx.Amount4.75 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 atTemperature70 °C for Duration00:10:00 in lysis buffer / Lammeli sample buffer mix

10m
Load Amount80 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 Duration01:00:00
For LRRK2 run gels Duration01:45:00

2h 45m
Transfer gels at 20V DurationOvernight at TemperatureRoom temperature

1h
Blot membranes
Blot membranes
2h 35m
Wash membranes in H2Or for Duration00:02:00 then 5 min in TBST

2m
Wash membranes in TBST for Duration00:05:00

5m
Incubate membrane in Ponceau S solution (Sigma P7170) for Duration00:05:00

5m
Rinse membrane in H2O and image
Wash 2 times in TBST for Duration00:05:00 each wash

5m
Incubate membrane in 5% milk in TBST (BioRad 1706404XTU) for Duration01:00:00 at TemperatureRoom temperature

1h
Wash 2 times in TBST for Duration00:02:00 each wash
2m
Incubate DurationOvernight at Temperature4 °C with primary antibody diluted in 5% milk in TBST

1h
ABCD
TargetSpeciesManufacturerDilution
pRab10rabbitAbcam ; ab2302611:500
Total Rab10rabbitCell Signaling ; 8127S1:1000
pRab8arabbitAbcam ; ab2302601:500
pRab12rabbitAbcam ; ab2564871:500
Total Rab12rabbitProtein Tech ; 18843-1-AP1:1000
GAPDHmouseProtein Tech ; 60004-1-Ig1:5000
ActinmouseMilipore; MAB15011:2000
LRRK2rabbitAbcam ; ab1334741:1000
p935 LRRK2rabbitAbcam ; ab1334501:1000
p1292 LRRK2rabbitAbcam ; ab2031811:1000
Primary antibodies for Rab proteins and LRRK2
Wash 3 times in TBST for Duration00:05:00

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

ABC
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 Duration00:05:00 and once in TBS

5m
Wash membrane in TBS for Duration00:05:00

5m
Image using ECL reagents (Amersham)
use Amount500 µL of solution A and Amount500 µL solution B for each membrane

Develop for at least Duration00:01:00 before imaging

1m