Abstract
With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10°C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 5 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the next positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).
| Original language | English |
|---|---|
| Pages (from-to) | 41-48 |
| Number of pages | 8 |
| Journal | Journal of Biochemical and Biophysical Methods |
| Volume | 15 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1987 |
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Keywords
- Alkaline pH range
- Immobilized pH gradien
- Isoelectric focusing
ASJC Scopus subject areas
- Biochemistry
- Biophysics
Cite this
Isoelectric focusing in immobilized pH gradients in the pH 10-11 range. / Gelfi, Cecilia; Bossi, Maria Lusia; Bjellqvist, Bengt; Righetti, Pier Giorgio.
In: Journal of Biochemical and Biophysical Methods, Vol. 15, No. 1, 1987, p. 41-48.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Isoelectric focusing in immobilized pH gradients in the pH 10-11 range
AU - Gelfi, Cecilia
AU - Bossi, Maria Lusia
AU - Bjellqvist, Bengt
AU - Righetti, Pier Giorgio
PY - 1987
Y1 - 1987
N2 - With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10°C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 5 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the next positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).
AB - With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10°C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 5 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the next positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).
KW - Alkaline pH range
KW - Immobilized pH gradien
KW - Isoelectric focusing
UR - http://www.scopus.com/inward/record.url?scp=0023424518&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023424518&partnerID=8YFLogxK
U2 - 10.1016/0165-022X(87)90061-3
DO - 10.1016/0165-022X(87)90061-3
M3 - Article
VL - 15
SP - 41
EP - 48
JO - Journal of Biochemical and Biophysical Methods
JF - Journal of Biochemical and Biophysical Methods
SN - 0165-022X
IS - 1
ER -