Biology Reference
In-Depth Information
4. Notes
1. A total of 10
7
cells were used for subcellular fractionation. Only
Fractions F1, F2, and F3 were used for further analysis.
2. The RC DC (reducing agent and detergent compatible) pro-
tein assay is a colorimetric assay for protein determination in
the presence of reducing agents and detergents. It is compatible
with a broader range of reagents allowing direct simplifi ed pro-
tein quantitation in complex sample solutions.
3. This is an alkaline copper tartrate solution.
4. This is a dilute Folin reagent.
5. This is a surfactant solution.
6. The DMF must be high quality and anhydrous, and every
effort should be taken to ensure it is not contaminated with
water. DMF, once opened, will start to degrade generating
amine compounds. Amine will react with the CyDye DIGE
fl uor minimal dyes reducing the concentration of dye available
for protein labeling.
7. The 1-mL syringe is used to take the DMF out of the fl ask
without opening it to avoid contamination with water.
8. The rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v)
CHAPS) ought to be made up without the DTT and ampho-
lytes and frozen at −20°C in 1 mL aliquots. Each aliquot will
then require 0.5% of carrier ampholytes and 50 mM DTT or
1.2% (v/v) of DeStreak to be added before being used.
9. Carrier ampholytes focusing will take place at their isoelectric
points and facilitate proteins focusing. DTT will reduce disul-
fi de bridges.
10. Unspecifi c oxidation of protein thiol groups occurs during 2D
electrophoresis of proteins, especially at pH > 7. In the result-
ing protein map, this is seen as horizontal streaking and extra
spots. Transforming the thiol groups into a stable disulfi de
using DeStreak reagent prevents unspecifi c oxidation. The
preparation of Immobiline DryStrip with DeStreak reagent
will result in 2D maps with reduced streaking between spots in
the pH range 7-9. It will also simplify the spot pattern as it
reduces the number of spots caused by oxidation of proteins.
At pH > 7, the isoelectric point of proteins may change due to
the transfer of thiols or oxidation products to disulfi des.
11. Iodoacetamide has to be freshly prepared and kept in darkness
in order to avoid oxidation to the corresponding carboxylic
acid. The cysteine residues previously reduced are alkylated by
carbamidomethylation, thus preventing the reformation of dis-
ulfi de bridges.
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