Extract preparations from cells and tissues

Authors

Piera Cilli

Procedure

The human gastric adenocarcinoma (AGS) cell line was grown in Dulbecco's modified Eagle's medium (DMEM/F-12) supplemented with 10% fetal bovine serum, 100U/ml penicillin, 100mg/ml streptomycin, in a humidified incubator with 5% CO2 at 37 °C. Near-confluent cultures were washed three times with ice-cold phosphate-buffered saline solution, harvested and resuspended at 106 cells/20ml in 10mM TrisHCl pH7.8, 200mM KCl buffer. Cell extracts were prepared by three freeze/thaw cycles according to ref. (31). After addition of an equal volume of 10mM Tris-HCl pH7.8, 200mM KCl, 2mM EDTA buffer containing 40% glycerol, 0.2% NP-40, 2mM DTT, 0.5mM PMSF and a cocktail of protease inhibitors (Complete mini, Roche), the cell suspension was rocked at 4°C for 1h and subsequently centrifuged at 16,000 rpm and 4°C for 10 min. The supernatant was recovered, dialyzed with 50mM Tris-HCl pH7.5, 50mM KCl, 0.1mM EDTA buffer containing 0.1mg/ml BSA, and 0.01% NP-40 and stored in small aliquots at -80°C. Striatum, motor cortex and cerebellum were dissected from 8- and 12- week-old R6/2 transgenic and wild-type mice. Briefly proteins were extracted by mechanical homogenization in a lysis buffer (50mM TrisHCl pH 7.4, 150mM NaCl, 1mM EDTA pH 8, 0.25% sodium deoxycholate, 1mM NaF, 1mM DTT, 0.5mM PMSF, 1% NP-40) containing a cocktail of protease inhibitors (Complete mini, Roche). Protein concentrations were determined using a Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA).

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Comments

• Cilli Piera Friday, 04 September 2015 - 14:49 UTC

  Ref 31:
  Biade S, Sobol RW, Wilson SH and Matsumoto Y. (1998). J. Biol. Chem., 273, 898-902

• 

  Alen Piljić Wednesday, 02 September 2015 - 22:29 UTC

  Hi Cilli,
  Can you add ref 31 which is mentioned in the protocol?
  Alen