Lysis buffer for protein extraction contained 150 mM NaCl, 1 mM EDTA, 25 mM Tris-HCl pH 8.0, 1% NP-40, 1 mM Na3VO4, 1 mM PMSF and 1 mM NaF. For lysis of cells, protease inhibitor cocktail (Sigma-Aldrich) was included in the lysis buffer. For lysis of tissue, 1 μg/mL pepstatin A, 1 μg/mL aprotinin and 1 μg/mL leupeptin were added to the lysis buffer. A polytron homogenizer (VWR International, Amsterdam, The Netherlands) was used to homogenize tissue samples. For the analysis of poly-ubiquitin chains, the deubiquitinase inhibitor [45 (link)] N-ethylmaleimide (50 mM; Sigma-Aldrich) was added to the lysis buffer. The BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine protein concentrations.
Polytron homogenizer
Manufactured by Avantor
Sourced in Netherlands
The Polytron homogenizer is a laboratory equipment used for dispersing, emulsifying, and homogenizing various types of samples. It functions by rapidly rotating a shaft with an attached rotor-stator system to create a high-shear force that effectively breaks down and mixes the sample material.
Automatically generated - may contain errors
2 protocols using polytron homogenizer
1
Protein Extraction from Cells and Tissues
2
Protein Extraction and Quantification from Murine Lung Tissue
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Protein extraction was performed with lysis buffer containing 150 mM NaCl, 1 mM EDTA, 25 mM Tris-HCl pH 8.0, 1% NP-40, 1 mM Na 3 VO 4 , 1 mM PMSF, and 1 mM NaF. For cell lysis, a protease inhibitor cocktail (Sigma-Aldrich) was added to the lysis buffer. For tissue lysis, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A were included in the lysis buffer, and tissue samples were homogenized using a polytron homogenizer (VWR International, Amsterdam, Netherlands). Protein concentrations were determined by the BCA assay (Thermo Fisher Scientific, Waltham, MS, USA). OTUB1 protein data from murine lung tissue were obtained from the LungMAP consortium (https://lungm ap.net/; 1U01HL122638; downloaded on 04/12/2020; Figure 3A).
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