Immobilized pH gradients: new pK values of acrylamido buffers in poly(N-acryloylaminoethoxyethanol) matrices.
A novel matrix consisting of N-acryloylaminoethoxyethanol, a hydrophilic monomer extremely resistant to hydrolysis, was recently reported by Chiari et al. (Electrophoresis, 1994, 15, 177-186). When using it as a matrix for grafting immobilized pH gradients for isoelectric focusing, a shift in protein spot position was noticed. This was attributed to a shift in pK values of the Immobiline buffers when changing from the standard poly(acrylamide) to a poly(N-acryloylaminoethoxyethanol) matrix. A series of 1 pH unit gradients was constructed, where a single buffering Immobiline was used and titrated with a counterion having a pK removed by at least 3 pH units from the nearest extreme of the generated pH interval. It was noted that all compounds became weaker acids and bases, with a delta pK ranging from -0.02 to -0.06 for the acids (pK 3.6, 4.4, and 4.6) and a delta pK ranging from -0.12 tp -0.20 for the bases. The new pK values for the seven commercially available buffers are thus pK 3.6-->pK 3.58, pK 4.4-->pK 4.36, pK 4.51-->4.45, pK 6.21-->6.09, pK 7.06-->6.94, pK 8.50-->pK 8.37, and pK 9.59-->pK 9.39. These values refer to 10 degrees C in the gel phase, the first value in poly(acrylamide) and the second in poly(N-acryloylaminoethoxyethanol).
Bossi
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Chair of Biochemistry, Faculty of Pharmacy, University of Milano, Italy.
Righetti
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PG
Chiari
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D000178:Acrylamides; D000180:Acrylic Resins; D000328:Adult; D002021:Buffers; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification; D006441:Hemoglobin A / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing / Q000379:methods*; D007700:Kinetics; D010047:Ovalbumin / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D015203:Reproducibility of Results; D014357:Trypsin / Q000302:isolation & purification
D000178:Acrylamides; D000180:Acrylic Resins; D002021:Buffers; D007202:Indicators and Reagents; D011506:Proteins; C089575:poly(N-acryloylaminoethoxyethanol); D010047:Ovalbumin; D006441:Hemoglobin A; D005087:Glucan 1,4-alpha-Glucosidase; D014357:Trypsin