Author: de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge
Title: NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells Document date: 2013_3_13
ID: 0jx6mwiw_20
Snippet: For cellular protein extraction, 1610 6 transfected HeLa cells were washed with PBS, and cell pellets were resuspended in 0.5 ml of lysis buffer (50 mM Tris-Cl, pH 7.5, 1% NP-40, 1% Nadeoxycholate, 0.1% SDS, 2 mM EDTA, 0.5 M NaCl, and 1/500 of SIGMA-protease inhibitor cocktail for mammalian cells). Preparations were incubated for 5 min in ice and then were passed through a narrow gauge needle (21G) to shear DNA. Then the lysates were cleared by c.....
Document: For cellular protein extraction, 1610 6 transfected HeLa cells were washed with PBS, and cell pellets were resuspended in 0.5 ml of lysis buffer (50 mM Tris-Cl, pH 7.5, 1% NP-40, 1% Nadeoxycholate, 0.1% SDS, 2 mM EDTA, 0.5 M NaCl, and 1/500 of SIGMA-protease inhibitor cocktail for mammalian cells). Preparations were incubated for 5 min in ice and then were passed through a narrow gauge needle (21G) to shear DNA. Then the lysates were cleared by centrifugation (14.000 g, 10 min at 4uC). Supernatants were used as fractions with the extracted proteins. MicroBCA Protein Assay Kit (Pierce) was used to determine protein concentrations. Western blot analysis was performed as described [36] , but using 60 mg aliquots of extracted proteins and specific anti-Flag (SIGMA) or anti-eGFP (Abcam) primary antibodies.
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