Biology Reference
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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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