Western blot (NuPAGE and MES buffer)
Laura Smith
Abstract
Cells were lysed in 1% Triton X 100 lysis buffer with protease and phosphatase inhibitors. Cell lysates were electrophoresed with NuPage™ Bis-Tris protein gels. Proteins were transferred to a PVDF membrane, blocked in 10% milk and treated with primary and secondary antibodies in 5% milk. Antibody binding was detected using the GE Healthcare Amersham™ electro-chemi-luminescence (ECL)™ Prime Western Blotting Detection Reagent.
Steps
Gel Electrophoresis
Make loading dye using NuPAGE Sample Buffer (4X) and Reducing agent (10X). For 18 µL of sample, add 6 µL sample buffer and 1 µL reducing agent.
Combine sample and loading dye and heat at 70 °C for 10 minutes .
Prepare precast gel (NuPAGE) by removing comb and the tape at the bottom of the gel chamber.
Set up the gel chamber in the gel tank so that the well openings of the precast are facing inwards toward each other.
Fill the gel chamber with 250 mL((1X) NuPAGE MES running buffer.
Remove samples from heat and spin at 10K for 1 minute to collect any evaporated sample in bottom of tube.
Load samples into gel using hamilton pipette tips. Place protein ladder marker in the first or last well.
Run gel at 200V for 45 minutes or until the protein has migrated far enough into the gel.
Transfer
Prepare Towin transfer buffer: 25 mM Tris, 192 mM glycine, pH 8.3, with 20% methanol (vol/vol).
To prepare 1 L of buffer:
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800 mL distilled H2O
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200 mL methanol
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3.03 g Tris base
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14.4 g Glycine
Soak PVDF membrane in methanol.
Soak transfer sandwich components (2 sheets of filter paper and 2 pads) in Towin transfer buffer until completely damp. Use hands or roller to remove any air bubbles.
Make up transfer sandwich in casette being careful to avoid trapping any air between layers. Layers should be 2 pads, 1 filter paper, gel, PVDF membrane, 1 filter paper and 2 pads.
Place cassette in transfer tank with transfer buffer and run for 1 hour at 30V.
Blocking and immunoblotting
Remove membrane from sandwich, noting the orientation relative to the gel to prepare for blocking.
For analysis of alpha-synuclein, fix the membrane with 0.4% PFA and 0.01% (v/v) glutaraldehyde for 30 minutes prior to blocking.
Block membrane in 10% semi-skimmed milk in PBS at room temperature for 1 hour.
Add desired primary antibody in 5% semi-skimmed milk in PBS and incubate with membrane for 1 hour at room temperature or 4°C over-night.
Wash membrane twice in 0.4% PBS-Tween.
Add appropriate HRP-linked secondary antibodies to 5% semi-skimmed milk (1:250) and incubate with membrane for. hour at room temperature.
Wash membrane three times in 0.4% PBS-Tween. Wash once in PBS.
Incubate membrane with 2 mL GE Healthcare Amersham™ electro-chemi-luminescence (ECL)™ Prime Western Blotting Detection Reagent for 5 minutes and drain excess reagent.
Develop blots using the Bio-Rad ChemiDoc imaging system (Bio-Rad) using the ‘chemi’ protocol with signal accumulation mode, calibrated to the signal intensities.
