Biology Reference
In-Depth Information
6. MOPS buffer (25×): Dissolve 209.26 g of MOPS in deionized water and fill to
1 L. Adjust the pH to 7.0 and autoclave buffer for 10 min.
7. l-Amino acid mixtures (50×): Prepare mixtures of one to four different amino
acids: (1) alanine, valine, leucine, isoleucine; (2) aspartate, glutamate; (3) serine,
threonine, cysteine; (4) arginine, lysine, proline; (5) phenylalanine, tryptophane,
histidine; (6) glycine. With the exception of glycine, dissolve 0.8 g of each amino
acid in 100 mL of deionized water. In case of glycine, use 8 g. It is important
to dissolve the amino acids of one mixture successively. Sterilize mixtures by
filtration (0.45 μm).
8. Vitamins (1000×): Prepare all vitamin solutions separately—0.036 m
M
cyanocobalmine, 0.29 m
Mp
-aminobenzoate, 0.04 m
M
biotin, 0.81 m
M
nicotinic
acid, 0.21 m
M
Ca-d-pantothenic acid, 0.62 m
M
pyridoxamine hydrochloride,
0.29 m
M
thiaminium dichloride, and 0.26 m
M
riboflavin. Dissolve the vitamins
in deionized water and sterilize by filtration (0.45 μm).
9. Trace elements (1000×): All trace element solutions are prepared separately—
0.51 m
M
ZnCl
2
, 0.5 m
M
MnCl
2
, 0.097 m
M
H
3
BO
3
, 1.46 m
M
CoCl
2
, 0.015
m
M
CuCl
2
, 0.1 m
M
NiCl
2
, and 0.148 m
M
Na
2
MoO
4
. Dissolve trace elements
in deionized water and sterilize by filtration (0.45 μm).
10. 0.5 m
M
FeCl
2
: Dissolved in 2
N
HCl.
11. 2
N
NaOH in deionized water.
12. TE Buffer: 10 m
M
Tris, 1 m
M
EDTA, pH 7.5, autoclaved.
13. Glass beads (0.10-0.11 mm, Braun, Melsungen, Germany).
14. Ribolyser (Thermo Electron Cooperation, Waltham, USA).
15. 100% (w/v) trichloroacetic acid (TCA) solution: Dissolve TCA in deionized
water and prepare fresh.
16. 8
M
Urea/2
M
Thiourea solution: Dissolve 2.4 g of urea and 0.76 g of thiourea
in 1 mL of water (deionized water additionally purified by using the water
purification system “Synergy” from Millipore, Schwalbach, Germany) and bring
to 5 mL with the same water. Store aliquots at -20 °C.
17. Stop solution: 15 mg of l-methionine and 10 mg of chloramphenicol dissolved
in 10 mL 0.1
M
Tris-HCl (pH 7.5).
2.2. Difference Gel Electrophoresis Labeling of Proteins
1. 99.8% anhydrous dimethylformamide (DMF).
2. 10 m
M
l-lysine in deionized water.
3. CyDye Difference Gel Electrophoresis (DIGE) Fluors for Ettan DIGE (GE-
Healthcare, Little Chalfont, UK): The dye tubes (Cy2, Cy3, Cy5) should be
allowed to warm to room temperature for 5 min. Add 10 μL DMF (freshly opened)
to each dye tube and mix. The tubes now contain 1 m
M
Cy2, Cy3, or Cy5 dye in
DMF and should be vortexed vigorously for 30 s. Afterward, centrifuge the tubes
for 30 s at 12,000 ×
g
. The fluorochrome can now be used. Unused CyDye stock
solutions should be returned to -20 °C as soon as possible and stored in the dark.
4. pH indicator strips (pH 4.5-10.0).
5. 50 m
M
NaOH in deionized water.
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