Western Blot Protocol: Difference between revisions
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*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] [http://products.invitrogen.com/ivgn/product/LC5925 standard] into first and 5th lane | *Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] [http://products.invitrogen.com/ivgn/product/LC5925 standard] into first and 5th lane | ||
**Mini-PROTEAN TGX Precast gels can only handle up to 30 μL of total solution | **[[Mini-PROTEAN TGX]] Precast gels can only handle up to 30 μL of total solution | ||
**Mini-PROTEAN TGX Precast gels have 10 wells, plan accordingly | **[[Mini-PROTEAN TGX]] Precast gels have 10 wells, plan accordingly | ||
*Load 25 μL sample into each gel well | *Load 25 μL sample into each gel well | ||
*record which lane contains which sample | *record which lane contains which sample | ||
| Line 115: | Line 115: | ||
*Put gel in TB for 10 min | *Put gel in TB for 10 min | ||
*Use Immun-Blot PVDF Membrane [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=162-0174 #162-0174] | |||
*Put new membrane in 100% MeOH | *Put new membrane in 100% MeOH | ||
**leave in MeOH until turns opaque | **leave in MeOH until turns opaque | ||
Revision as of 16:27, 15 May 2013
WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN
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 ?
- 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
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- Make Running Buffer (RB)
RUNNING BUFFER (RB)
- Prepare Mini-PROTEAN Tetra Cell tank for electrophoresis (see video)
- Use these gels if doing electrophoresis on the Mini-PROTEAN rig: 4–20% Mini-PROTEAN TGX Precast Gel #456-1093
- 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 first and 5th lane
- Mini-PROTEAN TGX Precast gels can only handle up to 30 μL of total solution
- Mini-PROTEAN TGX Precast gels have 10 wells, plan accordingly
- Load 25 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
4. BLOT TRANSFER (GEL TO MEMBRANE)
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BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS
- Make transfer buffer (TB)
TRANSFER BUFFER (TB)
- Put gel in TB for 10 min
- Use Immun-Blot PVDF Membrane #162-0174
- 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
- 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
PBS-T-Block DILUTION METHOD
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
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!