Rate of ERAD of misfolded proteins in Saccharomyces cerevisiae

Authors：

Category

Biosynthesis & Metabolism

Protocol Name

Authors

Hosomi, Akira
Glycometabolome Team, RIKEN Advanced Science Institute

KeyWords

Reagents

●	Liquid Medium: Yeast Nitrogen base (w/o amino acids) 0.67%, Glucose 2%, Amino acids and pyrimidines 20–50 μg/mL
●	Sample Buffer: 0.0625 M Tris-HCl (pH 6.8), 5% β-mercaptoethanol, 2% SDS, 5% Sucrose, 0.04% bromophenol blue
●	Dimethyl sulfoxide (DMSO)
●	100 mg/mL Cycloheximide (CHX) in DMSO
●	1 M Sodium azide (NaN₃) in distilled water

Methods

Rate of ERAD of misfolded proteins in Saccharomyces cerevisiae

1)	Yeast cells were grown at optimum temperature for overnight in the 1 mL liquid medium.	Comment 0

2)	The cells were grown at to OD₆₀₀=~2.0 at optimum temperature for 4 to 6 h in the 5 mL of liquid medium.	Comment 0

3)	The cells can be incubated at the restrictive temperature if needed.	Comment 0

4)	12.5 μL of DMSO or CHX (100 mg/ mL) are added to the cell culture (final concentration of 0.25 mg/mL).	Comment 0

5)	At the appropriate time points, 1 mL cell cultures were collected into a new tube that contains 20 μL of 1 M NaN₃.	Comment 0

6)	The cells are harvested by centrifuge at 3,000 × g for 1 min.	Comment 0

7)	Discard the supernatant and resuspend 720 μL of distilled water.	Comment 0

8)	Add 80 μL of 1 M NaOH and mix by vortex.	Comment 0

9)	Incubate for 10 min at room temperature.	Comment 0

10)	The cells are collected by centrifuge at 3,000 × g for 1 min.	Comment 0

11)	The cells are resuspended in the sample buffer at a cell concentration of (100 OD₆₀₀/mL) and mix by vortes.	Comment 0

12)	Heat the samples at 95°C for 5 min.	Comment 0

13)	Remove the cell debris by centrifuge at 10,000 × g for 3 min.	Comment 0

14)

Analyze the supernatants by SDS-PAGE, followed by western blotting with appropriate antibodies.

Comment 0

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Date of registration：2014-12-04 16:46:53