TITLE: Western blot (NuPAGE and MES buffer)
AUTHORS: Laura  Smith

[DESCRIPTION]
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]
SECTION: Gel Electrophoresis
1. 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.

SECTION: Gel Electrophoresis
2. Combine sample and loading dye and heat at 70 °C  for 10 minutes . 
SECTION: Gel Electrophoresis
3. Prepare precast gel (NuPAGE) by removing comb and the tape at the bottom of the gel chamber. 
SECTION: Gel Electrophoresis
4. Set up the gel chamber in the gel tank so that the well openings of the precast are facing inwards toward each other. 
SECTION: Gel Electrophoresis
5. Fill the gel chamber with 250 mL((1X) NuPAGE MES running buffer.
SECTION: Gel Electrophoresis
6. Remove samples from heat and spin at 10K for 1 minute to collect any evaporated sample in bottom of tube.
SECTION: Gel Electrophoresis
7. Load samples into gel using hamilton pipette tips. Place protein ladder marker in the first or last well.

SECTION: Gel Electrophoresis
8. Run gel at 200V for 45 minutes or until the protein has migrated far enough into the gel. 
SECTION: Transfer
9. Prepare Towin transfer buffer: 25 mM Tris, 192 mM glycine, pH 8.3, with 20% methanol (vol/vol).
To prepare 1 L of buffer:
- 800 mL distilled H2O
- 200 mL methanol
- 3.03 g Tris base
- 14.4 g Glycine

SECTION: Transfer
10. Soak PVDF membrane in methanol. 

SECTION: Transfer
11. 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. 
SECTION: Transfer
12. 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.

SECTION: Transfer
13. Place cassette in transfer tank with transfer buffer and run for 1 hour at 30V. 
SECTION: Blocking and immunoblotting
14. Remove membrane from sandwich, noting the orientation relative to the gel to prepare for blocking.
SECTION: Blocking and immunoblotting
15. Block membrane in 10% semi-skimmed milk in PBS at room temperature for 1 hour.
SECTION: Blocking and immunoblotting
16. 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.
SECTION: Blocking and immunoblotting
17. Wash membrane twice in 0.4% PBS-Tween.
SECTION: Blocking and immunoblotting
18. Add appropriate  HRP-linked secondary antibodies to 5% semi-skimmed milk (1:250) and incubate with membrane for. hour at room temperature.
SECTION: Blocking and immunoblotting
19. Wash membrane three times in 0.4% PBS-Tween. Wash once in PBS.
SECTION: Blocking and immunoblotting
20. Incubate membrane with 2 mL GE Healthcare Amersham™ electro-chemi-luminescence (ECL)™ Prime Western Blotting Detection Reagent for 5 minutes and drain excess reagent.


SECTION: Blocking and immunoblotting
21. Develop blots using the Bio-Rad ChemiDoc imaging system (Bio-Rad) using the ‘chemi’ protocol with signal accumulation mode, calibrated to the signal intensities. 
SECTION: Blocking and immunoblotting
14.1. For analysis of alpha-synuclein, fix the membrane with 0.4% PFA and 0.01% (v/v) glutaraldehyde for 30 minutes prior to blocking.