Protein extraction from chondrocytes

Lysis buffer

25 mM Tris-HCl pH 7.4

150 mM NaCl

1 mM EDTA

1% Triton X-100

5% Glycerol

Note: store at 4°C, add protease inhibitors (0.5mM PMSF) before use

Protocol

resuspend the cell pellet in 1.2 ml of PBS and count the number of cells/ml

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prepare aliquots of 105 cells in 1 ml

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centrifuge the aliquots at 9500 x g for 10 min

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resuspend pellets in 25µl of SDS-PAGE buffer with DTT (15µl dH2O, 5µl of 5 x SDS-PAGE buffer, 5µl 1M DTT)

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incubate at 95°C for 10 min

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freeze at -20°C until needed

Modification

Protein isolation with cross-linking and non-reducing conditions

resuspend pellet in 2 ml of cross-linking agent (25 mM NEM in PBS, store at 4°C)

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incubate on ice for 20 min

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centrifuge at 1000 rpm for 5 min

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wash with PBS

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centrifuge at 1000 rpm for 5 min

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incubate in lysis buffer containing protease inhibitors on ice for 5 min, mixing gently

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centrifuge at 13000 rpm for 10 min

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transfer supernatant to new tubes
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store at at -80°C