1. Proper washing of the pellet with lipid removal wash after derivatization is crucial to reduce the background signal on the blot due to excess DNPH. In addition, proper washing removes lipids.
2. Remove the ethanol and ethylacetate solution completely before adding the rehydration buffer, as excess of this mixture could lead to precipitation of proteins.
3. The addition of mineral oil will prevent sample evaporation.
4. Paper wicks will prevent IPG strips from burning.
5. Cover the equilibration tray with polyethylene sheets and wrap the equilibration tray with aluminum foil to prevent moisture formation.
6. Commercially available agarose solution comes with bromophenol blue dye that can be used as a tracking dye to monitor the electrophoresis.
7. DTT can be used to break disulfide bonds.
8. IA can be used to stabilize the disulfide bond once it is cleaved by DTT.
9. Washing is not required between blocking and primary antibody treatment.
10. One tablet of Sigma Fast is dissolved in 10 mL of deionized water.
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