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- Cell lysis radioimmunoprecipitation assay (RIPA) buffer.
150 mM sodium chloride, 1.0% NP-40 or Triton X-100,
0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sul-
fate), 50 mM Tris, pH 8.0. - Protease and phosphatase inhibitors: 1 mM phenylmethylsul-
fonyl fluoride (PMSF), 5 mM ethylenediaminetetraacetic acid
(EDTA), 1 mM ethylene glycol tetraacetic acid(EGTA), and
5–10 mM sodium fluoride. - Bicinchoninic acid assay (BCA) protein assay kit.
- Protein loading buffer: 4% SDS, 10% 2-mercaptoethanol, 20%
glycerol, 0.004% bromophenol blue, 0.125 M Tris–HCl at
pH 6.8. - 30% acrylamide/Bis solution.
- 1.5 M Tris–HCl.
- 10% SDS.
- N,N,N′,N′-tetramethylethylene-diamine (TEMED).
- 10% Ammonium persulfate (APS).
- Gel electrophoresis system.
- Gel running buffer. (For a10× buffer, add 30.0 g of Tris base,
144.0 g of glycine, and 10.0 g of SDS in 1000 mL of
H 2 O. Dissolve all the ingredients by stirring. Adjust the pH of
the buffer to 8.3 dilute to 1× before use. - Protein standard ladder.
- Polyvinylidene fluoride (PVDF) membrane.
- Gel transfer buffer. For 1×, add 6.04 g Tris base, 28.8 g gly-
cine together in 1.6 L of double distilled water (ddH 2 O).
Then, add 200 mL methanol and mix. Finally, add ddH 2 O to
a final volume of 2 L. - Ponceau S solution.
- Blocking buffer: 5% dry milk powder.
- MET antibody and corresponding secondary antibody.
- Wash buffer: Tris-buffered saline and Tween 20 (TBST) buf-
fer or phosphate-buffered saline and Tween 20 (PBST). - Trans-blot gel transfer system.
- Chemiluminescent Western blot detection kit.
- Gel imaging systems.
3 Methods
- Grow the cells up to 70–80 confluency in complete media
(add 10% FBS and 1% penicillin and streptomycin in DMEM,
2 mM glutamine). - Wash cells with PBS and collect the cells after trypsinization.
2.2 Western Blot
Analysis
3.1 Transfection
of Cultured Cancer
Cells with MET shRNA
Lentiviral Particles
Farhadul Islam et al.