Western Blot Protocol

WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN

BRADLEY MONK

UCSD MOLECULAR COGNITION LAB

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1. TISSUE COLLECTION

• Anesthetize rodent with isofluorine (or equivalent) and decapitate
• Extract brain and flash-freeze in 2-methylbutane cooled to -20oC with dry ice
• Store brain at -80 in sucrose or OCT for no more than 1 week

2. TISSUE DISECTION & PREPARATION

• Prepare Homogenization Buffer Reagent (HBR) at 4 deg C
• HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA
• Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR

Each sample will require a total of 500 μL HBR

Why 500 μL ?

Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per brain, you will need to prep 3 mL HBR per brain

• Keep stock HBR and aliquots chilled in ice bath as much as possible.
• Aliquot 200 μL HBR (per section) into labeled vials.
• Prepare a freeze-mount station for slides
  • this can be done simply by placing dry ice below a flat piece of glass or plastic
• Place first slide onto freeze mount station to pre-cool
• Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC
  • (remember to mark one side of brain with pin if hemisphere identification is important, or only cut one hemisphere at a time)
• Collect target section on tip of soft-bristle brush
• Pipette 250 μL droplet of HBR onto slide
• Float tissue section onto this droplet and use brush to unfold
• Once flat, use scalpel to dissect out region of interest (ROI)
• Leave ROI on slide and remove unwanted tissue from slide
• Use pipette to suck-up 100 μL of HBR+ROI tissue
• Drop this 100 μL into the appropriate vial containing 200 μL HBR
• Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer
• Use centrifuge to spin samples at 1000*g for 15 min
• Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR
• Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL.
• Use centrifuge to spin samples again at 1000*g for 60 min
• Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR
• Store sample at -80 deg C for no more than 1 week

3. SDS-PAGE (GEL ELECTROPHORESIS)

SDS-PAGE STEP 1 -- PREP SAMPLE

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• Thaw samples to 4oC and re-homogenize
• Extract 30 μL from sample and drop in new vial
• Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)
• Heat at 95 deg C for 5 minutes
• Vortex while hot and place on ice

SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS

Make Running Buffer (RB)

RUNNING BUFFER (RB)

Make 1000 mL RB

• 950 mL diH2O
• 50 mL Tween-20

• Remove white tape from gel plastic
• Set up gel cassette in XCell mini-cell tank
  • use spacer if needed
• Remove comb from top of gel plastic

• Pour some RB into center gel compartment and look for leakage
• Finish filling center gel compartment above white scaffold
  • make sure RB fills all the gel wells
• Fill exterior compartment with RB
  • don’t overflow into center gel compartment

• Load SeeBlue+2 standard into first lane
• Load 40 μL sample into each gel well
  • record which lane contains which sample

• Run gel 200V/50 minutes -- or until
  • dye is at the end of the plate
  • target band is in the center of the gel

Stopping Point

If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C

4. BLOT TRANSFER (GEL TO MEMBRANE)

BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS

Make transfer buffer (TB)

TRANSFER BUFFER (TB)

Make 1000 mL TB

• 750 mL diH2O
• 200 mL MeOH
• 50 mL 20x NuPAGE TB

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• Put gel in TB for 10 min

• Put new membrane in 100% MeOH
  • leave in MeOH until turns opaque
  • usually less than < 20 sec
• Remove membrane from MeOH and put in TB
  • leave in for 10 min

BLOT TRANSFER STEP 2 -- PREP CASSETTE

Membrane Transfer Cassette

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• Place cassette, dark side down and fill tray with TB
• Roll out bubbles during each step
  • moisten everything going in cassette with TB
  • put a sponge in the cassette
  • put 2 Whattman papers on top of sponge
  • put the gel on top of Whattman papers
  • put the membrane on gel
  • put 2 Whattman papers on top membrane
  • put another sponge on top of Whattmans
• Close frame with light side on top and lock.

BLOT TRANSFER STEP 3 -- RUN TRANSFER

• Add TB to Tetra Cell tank until 2/3 full
• Place cassette in tank
  • lock should face upward
  • black side of cassette to black side of electrode insert
• Fill tank to the fill-line with TB
• Run 150V/1hr

5. ANTIBODY EXPOSURE

ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER

Make PBS-T Block Buffer (milk + PBS + Tween)

• Use 5 g Carnation Powder Milk per 100 mL PBS-T
  • PBS
  • Tween20
  • Carnation powder milk

PBS-T-Block QUICK METHOD

Make 1000 mL PBS-T

• 900 mL diH2O
• 100 mL PBS-10x
• 1 mL Tween-20

Make 100 mL PBS-T-Block

• 5 g Carnation
• 100 mL PBS-T

PBS-T-Block DILUTION METHOD

Make a 10% Tween solution

• 10 mL Tween20
• 90 mL PBS-1x

Make 1000 mL PBS-T

• 10 mL Tween-10%
• 950 mL PBS-1x

Make 100 mL PBS-T-Block

• 5 g Carnation
• 100 mL PBS-T

ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK

• Remove membrane from cassette
• Wash membrane with PBS-T 2X
• Place membrane in PBS-T-Block for 1hr.
• Wash membrane 2X/5min with PBS-T

ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION

• Make 1° Antibody in PBS-T-Block.
• Add membrane and incubate overnight at 4 deg C on rocker table.

• Wash 4X/ 5min. with PBS-T.
• Make 2° Antibody in PBS-T-Block. (Ms 1:200)
• Add membrane and incubate 1 hr.
• Wash 4X/5 min. with PBS-T.
• Wash 3X/ 5 min. with diH2O.

Stopping Point

Incubation overnight should be done in a cold room at 4o C

6. BLOT VISUALIZATION

BLOT VISUALIZATION STEP 1 -- PREP STATION

• Place saran wrap on top of upside down box tray
• Have another box ready to place on top
  • this protects membrane and Dura from light
• Place membrane on top of saran wrap

BLOT VISUALIZATION STEP 2 -- PREP DURA

• Pour Dura A in conical vial-A and Dura B in conical vial-B
  • use 4 mL total Dura per gel

• Mix Dura A with Dura B and pour onto membrane
  • do this quickly
  • use drop method so Dura doesn’t run off membrane
  • protect from light by putting box on top
• Incubate for 5 min
• After the 5 min incubation, visualize blot in ECL machine ASAP!