1. The thiol group can engage in redox reaction only when in the thiolated (deprotonated) state (-S-), which occurs when the pH of the solution > pKa value of the cysteine residue. Free cysteine has a pKa of 8.3, and cysteine residues have pKa values from 4 to 10 depending on their amino acid environment. Therefore cell lysis under acidic conditions, which is best achieved with TCA (pH < 1), will keep cysteine residues in their protonated form thus preventing any redox modifications.
2. During acetone washing, the protein pellet should be manually brought to a powder with the help of a spatula.
3. Denaturing buffer containing urea should not be used at a temperature greater than 30°C, which may change protein pI.
4. A low protein concentration can be the result of partial protein during the acetone wash or to low cells lysis efficiency.
5. Fluorescent dyes are of non-negligible sizes. When using these reagents, protein spot signals very often appear as doublets of slightly different molecular size (Fig.13.4), which can be accounted by an uneven number of modified thiols per protein. The use of dyes at concentrations that saturate pro-tein-thiols should circumvent this problem. However this is not possible due to the price of the reagents. We therefore recommend instead using a higher concentration of IAM.
6. Alkylation temperature at 4°C decreases the nonspecific fluorescent labeling.
7. The presence of bromophenol blue, which migrates towards the anode, allows monitoring the quality of protein sample migration during isoelectrofusing.
8. DTT is omitted during both first and second dimension to prevent subsequent dye labeling of any remaining oxidized thiols by the dyes that are not removed in this particular procedure.
9. Air bubbles under the IPG strip should be avoided, because they perturb Iso-electro-focusing.
10. The plastic side and not the gel side of the IPG strip must be placed against the glass plate. When placing the IPG strip on the upper edge of the SDS polyacrylamide gel, it is also recommended to avoid air bubbles.
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