Biomedical Engineering Reference
In-Depth Information
3. YM-30 fi lter-unit size 15 mL (Amicon Ultra centrifugal fi lter
units Ultra-15, MWCO 30 kDa; Millipore, Millford, MA).
4. YM-30 fi lter-unit size 0.5 mL (Amicon Ultra 0.5 mL centrifu-
gal fi lters, MWCO 30 kDa; Millipore).
5. YM-10 fi lter-unit size 15 mL (Amicon Ultra centrifugal fi lter
units Ultra-15, MWCO 10 kDa; Millipore).
6. YM-10 fi lter-unit size 0.5 mL (Amicon Ultra 0.5 mL centrifu-
gal fi lters, MWCO 10 kDa; Millipore).
7. 1 M Tris-HCl stock solution (pH7.8): Dissolve 121.44 g Tris
base in 800 mL deionized H
2
O and adjust pH to 7.8 with
HCl. Make up the volume to 1 L by adding deionized H
2
O
and remove debris by fi ltration using membrane fi lter (pore
size: 0.45
m).
8. 20 mM Tris-HCl (pH 7.8): Dilute 1 M Tris-HCl stock solu-
tion 50-fold with deionized H
2
O.
9. Vacuum dryer.
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1. Glass beads (GB-05; diameter 0.5 mm; TOMY, Tokyo, Japan).
2. Bead Smash 12 (Wakenyaku, Kyoto, Japan).
3. 20 mM Tris-HCl (pH 7.8) containing 0.1 % SDS.
2.6 Preparation
of Intracellular
Proteins
1. 0.5 M Tris-HCl stock solution (pH 6.8): Dissolve 60.72 g Tris
base in 800 mL deionized H
2
O and adjust pH to 6.8 with
HCl. Make up the volume to 1 L by adding deionized H
2
O.
2. 2× buffer for SDS-PAGE: Mix 2 mL of 0.5 M Tris-HCl
(pH6.8), 4 mL of 10 % SDS, 2 mL glycerol, and 1 mg bromo-
phenol blue. Make up the volume to 10 mL by adding deion-
ized H
2
O and store at −20 °C. Add 10 % volume of
mercaptoethanol before use.
3. Continuous polyacrylamide gel (5-20 %; 120 × 100 mm;
e-PAGEL; Atto, Tokyo, Japan).
4. 1× Running buffer: Dissolve 30.36 g Tris base and 14.4 g gly-
cine in 800 mL deionized H
2
Oand add 10 mL of 10 %
SDS. Make up the volume to 1 L by adding deionized H
2
O.
5. FLAG protein (48 kDa cleavage control protein; Novagen,
Inc., Madison, WI).
6. Rainbow molecular weight marker proteins (Full-Range
Rainbow Molecular Weight Markers, GE Healthcare,
Buckinghamshire, UK).
2.7 SDS-PAGE
1. Nitrocellulose membrane (0.45-
m pore size).
2. Anti-FLAG M2 antibody conjugated with horseradish peroxi-
dase (HRP; Sigma-Aldrich, St. Louis, MO).
3. Rabbit anti-baker's yeast phosphoglucose isomerase-1 (Pgi1)
antibody (Acris Antibodies GmbH, Hiddenhausen, Germany).
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2.8 Western Blotting
