Western Blot Protocol

From Bradwiki
Jump to: navigation, search

WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN


BRADLEY MONK

UCSD MOLECULAR COGNITION LAB



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

SDS-PAGE.png

If using the prescribed methods each sample should have 100 μL total volume
  • Pre-heat vial water bath to 85 deg C
  • Thaw samples to 4 deg C
  • Add 25 μL 4x RunBlue LDS Loading Buffer
  • (optional) Add 5% DTT (LIFE sciences)
  • Heat sample vials in the 85 deg C bath for 5 minutes
  • Vortex while hot - make sure membrane fraction is homogenized
  • Put vials in ice bath

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

OR

Make 1000 mL NuPAGE RB
  • 950 mL diH2O
  • 50 mL NuPAGE +MOPS


  • Remove green tape strip from bottom of gel cassette
  • Set up gel cassette in Mini-PROTEAN gel holder, wells facing inward, and put in tank
    • If running only 1 or 2 gels remove the 2nd companion gel-holder
  • Remove comb from top of gel cassette
  • Pour some RB into center gel compartment and look for leakage
  • Finish filling center gel compartment making sure RB covers wells
  • Fill exterior compartment with RB
  • Rinse wells with RB using pipette


  • Load SeeBlue+2 standard into 1st and 5th lanes
  • Load 25 μL sample into each gel well
  • record which lane contains which sample


  • Run gel 200V until marker dye runs off bottom of gel
    • approx. 30 min
    • ideally run until target band is in center of gel
  • After Electrophoresis, shut down system and remove cassette
  • Crack open cassette with notched (“well”) side of the cassette facing up
  • Remove and discard the top plate, allowing the gel to rest on the bottom (slotted) plate.
  • Continue to blotting stage without removing the gel from the bottom plate


Electrophoresis.png

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

Transfer Buffer.png


  • Put gel in TB for 10 min
  • Prepare an Immun-Blot PVDF Membrane #162-0174
    • Clip bottom right corner of membrane to help with identification
  • Soak PVDF membrane in 50 mL of 100% MeOH
    • 30 sec
  • Remove membrane from MeOH then soak in 50 mL TB
    • 10 min
  • Soak Whatman filter paper in 50 mL TB
    • 30 sec

BLOT TRANSFER STEP 2 -- PREP CASSETTE

Membrane Transfer Cassette

Membrane Cassette.png

  • 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
    • use razor to cut off top of gel (lane comb)
  • put the gel on top of Whattman papers
    • ladder-lane 1 to the left
  • 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.

Loading Cassette.png

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
  • put icepack inside tank, and put tank in ice bath
  • Fill tank to the fill-line with TB
  • Run 150V/1hr
After transfer, identify sample positions on membrane
  • turn over membrane so the clipped corner is on the bottom left
  • the blots are now facing upward
  • lane 1 ladder is now on the right
  • dip PVDF membrane in MeOH
    • (if stopping, dry membrane on filter paper overnight)

Blot Transfer.png

5. MEMBRANE BLOCK AND ANTIBODY INCUBATION

MEMBRANE BLOCK

Make PBST and PBSTb Block Buffer
  • PBST = PBS + Tween
  • PBSTb = PBS + Tween + Milk Block
  • Use 5 g Carnation Powder Milk per 100 mL PBST


PBST & PBSTb

Step 1 - Make 1000 mL PBST
  • 900 mL diH2O
  • 100 mL PBS-10x
  • 1 mL Tween-20


Step 2 - Make 100 mL PBSTb
  • 5 g Carnation
  • 100 mL PBST


Ready PVDF Membrane
  • Dip membrane in MeOH
  • Float membrane in PBST


Ready a Rocker Table for membrane blocking
  • Fill container with PBSTb
  • Put container on rocking table or shaker table
  • Put membrane in PBSTb container
    • Turn on rocker
  • Let incubate for 2 hours or overnight


ANTIBODY INCUBATION

Prepare for 1° Antibody Incubation

Wash off 1° Antibody

  • Dilute 1° Antibody in PBSTb
    • seal membrane with 8 mL of PBSTb + 1°
    • dilute 1° antibody 1000:1 in PBSTb
    • aka dilute 8 uL 1° in 8 mL PBSTb
  • Put membrane in plastic sealer bag
  • Seal 3 sides of plastic
  • Pour in 1° Antibody PBSTb mixture
  • Incubate for 24 h at 4° C on rocker table.
  • Fill 3 containers with PBST
  • Put membrane in 1st PBST wash
    • 5 min on rocker table
  • Put membrane in 2nd PBST wash
    • 5 min on rocker table
  • Rinse membrane in 3rd PBST wash



Prepare for 2° Antibody Incubation

Wash off 2° Antibody

  • Dilute 2° Antibody in PBSTb
    • Ms 1:200
    • seal membrane with 8 mL of PBSTb + 2°
    • dilute 2° antibody 1000:1 in PBSTb
    • aka dilute 8 uL 2° in 8 mL PBSTb
  • Put membrane in plastic sealer bag
  • Seal 3 sides of plastic
  • Pour in 2° Antibody PBSTb mixture
  • Incubate for 1 h at 4° C on rocker table.
  • Fill 3 containers with PBST
  • Put membrane in 1st PBST wash
    • 5 min on rocker table
  • Put membrane in 2nd PBST wash
    • 5 min on rocker table
  • Rinse membrane in 3rd PBST wash
  • Put membrane in diH2O wash
    • 5 min on rocker table
  • Put membrane in diH2O wash
    • 5 min on rocker table
  • Rinse membrane in diH2O




SeeBlue Novex.pngGluR234.jpg

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!


References & Notes

  1. The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.