Biomedical Engineering Reference
In-Depth Information
4. Anti-rabbit antibody conjugated to HRP (GE Healthcare).
5. Blocking solution (Blocking One; Nacalai Tesque, Inc., Kyoto,
Japan).
6. Can Get Signal
®
Immunoreaction Enhancer Solution
(Toyobo).
7. Stripping agent (WB Stripping Solution; Nacalai Tesque, Inc.).
8. ECL Plus Western blotting detection reagents (GE Healthcare)
(currently unavailable; we have tested ECL Prime from GE
Healthcare, but neither endogenous Pgi1p nor external con-
trol were detected by the methods described here. Please con-
sider changing the sample volume or using other detection
reagents such as Pierce Western Blotting Substrate Plus from
Pierce, Luminata series from Millipore, etc.).
9. ImageQuant LAS 4000 minisystem (GE Healthcare).
10. 1× PBS: Dilute 10× PBS (1,370 mM NaCl, 81 mM Na
2
HPO
4
,
26.8 mM KCl, 14.7 mM KH
2
PO
4
, pH 7.4, Sigma-Aldrich,)
tenfold with deionized H
2
O.
11. PBST: Add 0.1 % Tween 20 to 1× PBS.
12. Hybri-Bag (e.g., Hybri-Bag Hard, Cosmo Bio, Tokyo, Japan).
13. Heat Sealer (e.g., Poly Sealer, Cosmo Bio).
14. Seesaw-motion shaker.
15. Plastic tray (disposable).
3
Methods
To quantitatively evaluate the unconventional secretion of pro-
teins, the following procedures were performed: (1) Preparation of
plasmids, (2) Preparation of yeast cells producing recombinant
(tagged) proteins, (3) Detection and quantifi cation of extracellular
proteins by Western blotting, (4) Evaluation of unconventional
secretion by inhibiting conventional secretion pathway, and (5)
Determination of secretion-regulatory proteins using knockout
strains.
For every experiment, the yeast strain producing EGFP-FLAG
protein (i.e., yeast strain transformed with plasmid pUL-ATG-
EGFP) was used as a secretion-negative control. Both the EGFP
protein and FLAG-tag sequence do not contain signal sequences
for secretion; thus, detection of the EGFP-FLAG in culture media
of the negative control strain indicates that the proteins were arti-
fi cially “leaked” during the experimental procedure. This leak
often occurs due to careless experimental techniques such as inten-
sive mixing of cells during washes or rapid fi ltration of culture
supernatants before thoroughly eliminating cells by mild centrifu-
gation. If the negative control protein is detected in culture media,
