Current Gel Electrophoresis Approaches to Low-Abundance Protein Marker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 177

TABLE 1 Compatibility of CPLL Protein Elution Methods with 2-DE Analysis

Elution Method Eluate Treatment Prior to 2-DE Comments

Single elution with 10% sodium dodecyl
sulphate containing 50 mM DTT

Sodium dodecyl sulphate is not compatible
with the

Upon elimination of sodium dodecyl sulphate,
some proteins could precipitate, depending on
the nature of the initial extract.

￿

first dimension of 2-DE.

2 M thiourea-7 M urea-4% CHAPS-50 mM
cysteic acid.

Eluate is directly compatible with 2-DE
analysis.

Cysteic acid does not affect the isoelectric
migration phase.

￿

9 M urea-4% CHAPS-100 mM acetic acid, pH 3. What needs to be eliminated is acetic acid and

citric acid for pH gradients between 3 and 10.

Both could be directly used when the pH
gradient is narrow and starts above pH 4 e 5.

￿

9 M urea containing 2% CHAPS at pH 3.0 e 3.5
with 50 mM citric acid

￿

6 M guanidine-HCl, pH 6.0. Guanidine-HCl must be eliminated as

described above.

After elimination of guanidine, HCl proteins
are precipitated and dissolved in IEF
compatible solution.

￿

￿ 20 mM tris containing 7 M urea, 2 M thiourea

and 4% CHAPS pH 8.5.

Only for 2D-DIGE. These eluates are used
directly after the protein labeling with Cy-Dyes.

These eluates are not compatible with regular
2-DE.

￿ 20 mM sodium carbonate containing 7 M urea,

2 M thiourea and 4% CHAPS pH 8.5.

￿ 0.2 M glycine-HCl, 2% NP-40, pH 2.4 Proteins from these eluates are generally

cleaned by current precipitation processes
or other cleanup procedures.

These are partial eluents for sequential protein
desorption. Individually, they do not allow
eluting quantitatively captured proteins.

￿ 1 M acetic acid, 2% NP-40

￿ 1 M NaCl, 2% NP-40

0.1 M acetic acid containing 40% ethylene glycol

￿