Biology Reference
In-Depth Information
2. Cell subculturing:
Arabidopsis
cells (ecotype Columbia, Col0)
are subcultured every 10 days. 1/10 of the previous culture is
diluted in 250 mL of fresh MS medium. Cell culture is main-
tained at 23 °C under continuous light and shaking (127 rpm/
min) in an incubation shaker.
Check instructions
1. SDS stock solution: Prepare 250 mL of 20 % SDS solution
(288.38 g/mol) by dissolving 50 g of SDS in 190 mL of water).
Mix with stir bar/magnetic stirrer but do not vortex. Heat
(80 °C) the solution in the microwave to speed up the dissolv-
ing and bring the volume up to 250 mL with water. Store pref-
erentially the solution at 25 °C to avoid crystallization.
2.2 Cell Grinding
2. Hepes buffer: To prepare 1 L of 1 M Hepes (238.3 g/mol)
buffer, pH 7.7, add 238.3 g of Hepes powder to 850 mL of
water. Adjust the pH with NaOH pellets or saturated solution
at fi rst and use 2 M NaOH when pH gets close to the required
pH. Bring the volume up to 1 L with water.
3. EDTA solution: To prepare 0.5 L of a 0.5 M EDTA solution,
add 146.12 g of EDTA (292.24 g/mol) in 400 mL of water.
Adjust immediately to pH 8 with NaOH (add few NaOH pel-
lets at fi rst and use NaOH 2 M when pH gets close to the
required pH). Finally, bring the volume up to 500 mL with
water.
4. HENS buffer, pH 7.7: 150 mM Hepes, 5 mM EDTA, 0.5 mM
neocuproine 1 % SDS. To prepare 1 L of HENS buffer pH 7.7,
mix 150 mL of Hepes 1 M pH 7.7 (from stock) with 100 mL
of 0.5 M EDTA (from stock). Add 0.104 g of neocuproine
(208.269 g/mol) and 50 mL of 20 % SDS (from stock). Bring
the volume up to 1 L with water. Mix and verify the pH (7.7).
5. One Büchner funnel glass fi lter.
6. A blender (IKA Analytical mill A10—Fisher Scientifi c MPF-
400-010J) or a mortar (
see
Note 2
).
7. A 38
m sieve.
8. A vacuum pump and a funnel.
9. 1 L of acetone placed at −20 °C.
10. 50 mL centrifuge tubes.
11. A centrifuge with a rotor for 50 mL centrifuge tubes.
12. 2D Quant kit assay (GE healthcare 80-6483-56) (
see
Note 3
).
μ
1. MMTS stock solution: To prepare a 2 M MMTS solution, mix
212
2.3 BS-ICAT Labeling
L of MMTS (9.43 M is the initial concentration in the
bottle) and 788
μ
L of DMF. Prepare fresh and store at room
temperature in the dark until use.
μ
