Stable storage conditions of immobiline chemicals for isoelectric focusing

A range of zwitterionic acrylic acid derivatives, buffering in the neutral and basic pH ranges, have been synthesized by the Mannich reaction of malonic acid, formaldehyde and a secondary amine. These compounds include 2-(4-morpholinomethyl)propenoic acid pK2 7.59 +/- 0.03 (23 degrees C), 2-[bis(2-hydroxyethyl)aminomethyl]propenoic acid pK2 approximately 8.6 (20 degrees C), 2-[bis(2-hydroxypropyl) aminomethyl]propenoic acid PK2 approximately 8.7 (20 degrees C), 2-[N-(2-hydroxyethyl)-N-methylaminomethyl]propenoic acid pK2 9.22+/-0.08 (22 degrees C), 2-[N-ethyl-N-(2hydroxyethyl)aminomethyl]-propenoic acid pK2 approximately 9.6 (20 degrees C), and 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid, which has a sigmoidal buffering profile over the pH range 3-10. These zwitterionic acrylic acid buffers were successfully copolymerized with acrylamide to prepare immobilized pH gradients (IPGs) in the neutral to alkaline portion of the pH range. Bovine erythrocyte carbonic anhydrase isozymes were resolved on a pH 5-8 IPG prepared using 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid as the immobilized buffer, and horse heart myoglobin was focused on pH 7.1-8.1 and pH 7.5-7.7 IPGs, using 2-(4-morpholinomethyl)propenoic acid as the immobilized buffer. In both cases the pK 9.3 Immobiline compound was used as the strongly basic titrant. These new compounds, besides possessing more hydrophilic residues than the corresponding commercial basic acrylamido buffers (Immobilines), resist hydrolysis at alkaline pH values.

Section: Introductionmentioning

confidence: 97%

Synthesis of zwitterionic acrylic acid buffers for isoelectric focusing in immobilized pH gradients

Bellini

Manchester

1998

“…At present, no substitute for this titrant is in sight, although this compound is not quite suitable for our purposes. We are trying to synthesize a new tjtrant which, being a tertiary amine, will be fully soluble in n-propanol, which inhibits hydrolysis and autopolymerization ofbasic acrylamido buffers [12]. Notwithstanding all these problems, we can show that the use of these extended pH gradients is feasible and that the fractionation range for the 2-D methodology encompasses nearly 8.5 pH units.…”

Section: The Ph Gradientmentioning

confidence: 98%

Immobilized pH 2.5–11 gradients for two‐dimensional electrophoresis

Sinha

Köttgen

Westermeier³

et al. 1992

Self Cite

Extremely wide immobilized pH gradients, pH 2.5-11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two-dimensional map.

“…Comigration presents major problems when attempting to discern which protein in a given spot is responsible for changes in expression. Various solutions for achieving narrower pH gradients and higher resolution have been investigated to overcome this problem [4][5][6]. Also, gel run-to-run reproducibility is an issue since gels run differently under minor changes in experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

“…The method predominantly used to identify changes in cellular protein expression is two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) [1][2][3]. 2-D PAGE is capable of resolving typically 1000-2000 protein spots from whole cell lysates over a broad pH range (pH [3][4][5][6][7][8][9][10][11][12]. However, 2-D PAGE has several disadvantages for profiling protein expression owing to its limited resolution and reproducibility.…”

Section: Introductionmentioning

confidence: 99%

Narrow‐band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed‐phase separations

Zhu

Lubman

2004

Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.