Endoglycosidase digestion was performed as previously described35 (link) with a few modifications. Cell extracts from BY2 lines transformed with pTRA-CardB were prepared as previously described. Then, 3 μL of 10X concentrated denaturing buffer (500 mM sodium citrate, pH 5.5, 2% SDS, 10% β-mercaptoethanol) was added to 30 μL of cell extract and samples were boiled at 100 °C for 10 min. After boiling, samples were incubated for 5 min on ice and 3 μL of 10X G5 buffer (New England BioLabs, UK) were added to the samples together with 1.5 μL of 25X concentrated protease inhibitor cocktail (Roche, USA) and endoglycosidase H (2.5 mU) (Roche, USA). Samples were incubated at 37 °C overnight. Control samples were treated without endoglycosidase H. Finally, 0.25 volumes of 4X SDS-PAGE loading buffer were added to the samples and analyzed by SDS-PAGE and western blot as previously described.
Concentrated protease inhibitor cocktail
Manufactured by Roche
Sourced in United States
The 25X concentrated protease inhibitor cocktail is a laboratory reagent designed to inhibit the activity of proteases. It is a concentrated solution that can be diluted and added to samples to prevent the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using concentrated protease inhibitor cocktail
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Endoglycosidase Digestion of BY2 Extracts
2
Endoglycosidase Digestion of BY2 Cell Extracts
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Endoglycosidase digestion was performed according to 37 with a few modifications. Cell extracts from BY2 lines transformed with pTRA-CardB were prepared as previously described. Then, 3 L of 10X concentrated denaturing buffer (500 mM sodium citrate, pH 5.5, 2% SDS, 10% mercaptoethanol) was added to 30 L of cell extract and samples were boiled at 100 °C for 10 min. After boiling, samples were incubated for 5 min on ice and 3 L of 10X G5 buffer (New England BioLabs, UK) were added to the samples together with 1.5 L of 25X concentrated protease inhibitor cocktail (Roche, USA) and endoglycosidase H (2.5 mU) (Roche, USA). Samples were incubated at 37 °C overnight. Control samples were treated without endoglycosidase H. Finally, 0.25 volumes of 4X SDS-PAGE loading buffer was added to the samples and analyzed by SDS-PAGE and western blot as previously described.
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