Biology Reference
In-Depth Information
4
Notes
1. Mops-KOH (pH 7.6), EGTA (pH 8.0), and EDTA (pH 8.0)
should be prepared as 0.5 M stock solutions and stored at
4 °C. The pH of EGTA and EDTA should be adjusted using
NaOH. When BSA is dissolved, BSA powder should be preset
at room temperature. PMSF and SHAM should be prepared
as 1 and 1.6 M stock solutions in DMSO, respectively, and
stored at 4 °C. DTT should be stored at −30 °C as a 1 M stock
solution. PMSF, SHAM, and DTT should be diluted only as
needed just before use. If you prepare
Arabidopsis
PM frac-
tion, the homogenizing medium should consist of 0.5 M sor-
bitol, 50 mM Mops-KOH (pH 7.6), 5 mM EGTA (pH 8.0),
5 mM EDTA (pH 8.0), 1.5 % (w/v) polyvinylpyrrolidone-40
(molecular weight 40,000), 0.5 % (w/v) BSA, 2 mM PMSF,
4 mM SHAM, and 2.5 mM DTT.
2. KH
2
PO
4
/K
2
HPO
4
(K-P) buffer (pH 7.8) should be prepared
as a 0.5 M stock solution and diluted to make the MS-suspension
medium. First, 200 mL of 0.5 M K
2
HPO
4
and 30 mL of
0.5 M KH
2
PO
4
are prepared. The pH of the 0.5 M K
2
HPO
4
is adjusted to 7.8 by adding 0.5 M KH
2
PO
4
, monitored by a
pH meter. If you prepare
Arabidopsis
PM fraction, the MS
suspending medium should contain 10 mM KH
2
PO
4
/
K
2
HPO
4
(K-P) buffer (pH 7.8) and 0.3 M sucrose.
3. If you prepare
Arabidopsis
PM fraction, the fi nal concentration
of NaCl should be adjusted to 100 mM in the MS-suspension
medium.
4. Mops-KOH (pH 7.3) and EGTA (pH 8.0) should be pre-
pared as a 0.5 M stock solution and stored at 4 °C. If you
prepare
Arabidopsis
PM fraction, the PM suspending medium
consists of 10 mM Mops-KOH (pH 7.3), 1 mM EGTA (pH
8.0), and 0.3 M sucrose.
5. For
Arabidopsis
PM preparation, you should weigh 1.4 g of
polyethylene glycol 3350 and 1.4 g dextran and add to 9.4 mL
MS-suspension medium and 7.3 mL NaCl medium in a 40 mL
centrifuge tube.
6. When the pH of the Tris buffer is adjusted, the buffer solution
should be at room temperature. The pH of Tris can be affected
by the temperature of the solution. Addition of HCl results in
an increase in temperature by heat of neutralization and dilu-
tion. To avoid a temperature increase of the solution, add the
HCl slowly and intermittently.
7.
β
-mercaptoethanol is a reducing agent and should be added to
the sample buffer just before use.
