Transport of GPI anchored proteins from ER to Golgi ~Pulse-chase transport analysis of DAF from the ER to the Golgi

Authors：

Category

GPI anchored proteins

Protocol Name

Authors

Fujita, Morihisa
Research Institute for Microbial Diseases, Osaka University

Maeda, Yusuke
Research Institute for Microbial Diseases, Osaka University

Kinoshita, Taroh *
Research Institute for Microbial Diseases, Osaka University

*To whom correspondence should be addressed.

KeyWords

GPI anchor GPI-anchored proteins ER-to-Golgi transport glycosylphosphatidylinositol

Reagents

●	Cells highly expressing human DAF
●	Trypsin / EDTA
●	Phosphate-buffered saline (PBS)
●	Medium / 10% FCS
●	DMEM without L-methionine and L-cysteine (Gibco/Life Technologies, Carlsbad, CA 21013-024)
●	[³⁵S]-methionine/cysteine (example: EXPRE³⁵S³⁵S Protein Labeling Mix (Perkin Elmer, Waltham, MA NEG772))
●	Chasing cocktail stock (100×): 150 mM methionine and 120 mM cysteine 100 mM NaN₃
●	PBS containing 10 mM NaN₃
●	RIPA buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS)
●	Mouse anti-human DAF (IA10) (BD, Franklin Lakes, NJ)
●	Protein-A-Sepharose (or Protein-G-Sepharose)
●	TNET buffer (100 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM EDTA, 1% Triton X-100)

Instruments

●	Incubator (37˚C), centrifuge, rotator, fluorescent image analyzer (example: FLA-5100 (Fujifilm, Tokyo, Japan))

Methods

Pulse-chase analysis of DAF

1)	Harvest cells (2 × 10⁷).	Comment 1

2)	Centrifuge at 1400 rpm for 3min.	Comment 0

3)	Resuspend in 5.5 mL methionine- and cysteine-free DMEM / 10% FCS.	Comment 0

4)	Incubate for 30 min at 37˚C.	Comment 0

5)	Pulse with 55 μL (550 μCi) [³⁵S]-methionine/cysteine for 10 min.	Comment 0

6)	Add 1/100 volume of chasing cocktail (55 μL).	Comment 0

7)	Take up 1 mL cell suspension into new tube containing 100 μL of 100 mM NaN₃ at varying time points (0, 10, 20, 40, 60 min) and keep on ice.	Comment 0

8)	Wash with ice-cold PBS-containing 10 mM NaN₃.	Comment 0

9)	Lyse cells in 0.6 mL of RIPA buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitor cocktail for 30 min on ice.	Comment 0

10)	Centrifuge at 13,000 × g for 15 min at 4˚C.	Comment 0

11)	Aspirate supernatant into a fresh tube and rotate with 1.5 μg/tube monoclonal anti-DAF antibody (IA10) and 30 μL Protein-A-Sepharose. (Protein-A-Sepharose should pre-incubate with non-radiolabeled cell lysate for 1 h before starting immunoprecipitation to remove non-specific binders).	Comment 0

12)	Wash Protein-A-Sepharose with 1 mL TNET buffer 4 times.	Comment 0

13)	SDS-PAGE under reducing conditions.	Comment 1

14)

Analyze using a BAS 1000 analyzer.

Comment 0

Figure & Legends

Pulse-chase of DAF.

CHO-K1 cells stably transfected with human DAF were pulse-labeled with a mixture of [³⁵S]-methionine and -cysteine followed by chasing for the indicated times. DAF was immunoprecipitated using a monoclonal anti-DAF antibody, separated by SDS-PAGE and analyzed using a FLA-5100 analyzer.

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Date of registration：2015-01-07 14:25:31