Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 235

1% (w/v) CHAPSO ( see Note 3 ), protease inhibitor cocktail
1(Sigma).
4. Sucrose gradient solutions: MES buffer containing 10 mM
NaF, 2 mM Na 3 VO 4 , plus 90, 35 or 5% (w/v) sucrose ( see
Note 4 ).
5. Tris/urea buffer: 20 mM Tris, 8 M Urea, pH 7.4, 10 mM
NaF, 2 mM Na 3 VO 4 ,5mMDTT,0.2mMPMSF.
6. Mini glass homogenisers, 1 mL capacity (Jencons, Leighton
Buzzard, UK).
7. Ultra-Clear TM centrifuge tubes 14 mm

× 89 mm (Beckman

Coulter, Fullerton, CA, USA).
8. SW41 swing out rotor and buckets for ultra-centrifugation
(Beckman Coulter).

1. 4X Protogel Resolving buffer (National Diagnostics,
Atlanta, GA, USA): 1.5 M Tris-HCl. 0.4% SDS, pH 8.0.
2. Protogel Stacking buffer (National Diagnostics): 0.5 M
Tris-HCl, 0.4% SDS, pH 6.8.
3. Ultrapure Protogel protein sequencing grade acrylamide
stock solution (National Diagnostics): 30% (w/v) acry-
lamide, 0.8% (w/v) bis-acrylamide (37.5:1).
4. N, N, N', N' -Tetramethylethylenediamine for electrophore-
sis (TEMED, Sigma).
5. Ammonium persulphate 98% (Sigma): Freshly prepare a 10%
(w/v) solution in water for immediate use.
6. Modified 4X Laemmli buffer ( 7 ) : 125 mM Tris-HCl, pH
6.8, 40% (w/v) SDS, 20% glycerol, bromophenol blue 0.1%
(w/v). Add DTT to 10 mM concentration prior to use.
7. Tris-glycine 10X SDS running buffer (Invitrogen, Paisley,
UK).
8. 2-Propanol HPLC grade 99.5% (Sigma): Prepare in a glass
vial as a 50% solution in water.
9. SeeBlue R

2.3.SDS-PAGEfor
WesternBlot
Analysis

Plus2 prestained marker proteins (Invitrogen).

1. Wet transfer buffer: 25 mM Tris-HCl (do not adjust pH),
192 mM glycine, 20% v/v methanol.
2. Tris-buffered saline with Tween (TBS-T). Dilute 100 mL
10X stock TBS with 900 mL water and add 2 mL
Tween-20.
3. Nitrocellulose membrane 0.45

2.4.WesternBlotting
forFlotillin-1

μ m pore (Schleicher &

Scheull, Dassel, Germany).
4. Filter paper, extra thick 7.4

× 10 cm (Bio-Rad).

5. Sponge pads (Invitrogen).