Rapid purification of antibodies to single band purity using recycling isoelectric focusing

Binion, Steven B.; Rodkey, L. Scott; Egen, Ned B.; Bier, M.

doi:10.1002/elps.1150030507

Cited by 15 publications

(5 citation statements)

References 10 publications

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“…IEF is a high-resolution technique capable of resolving proteins that differ in pI by fractions of a pH unit (7,13). By this technique we obtained four fractions, and after subsequent gel filtration the P12 fraction, which reacted with anti-antigen A-2 serum by AGID and ELISA, was obtained.…”

Section: Discussionmentioning

confidence: 99%

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Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen

1992

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An internal protein was purified from cell extracts of BruceUa melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B.

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Section: Discussionmentioning

confidence: 99%

“…Preparative IEF. Preparative horizontal column IEF (7,12) was performed within a pH 4 to 6 ampholine (LKB) gradient by using a Rotophor Cell (Bio-Rad). CPE (0.3 mg/ml) was mixed with 2.5 ml of ampholines and brought to a total volume of 50 ml.…”

Section: Methodsmentioning

confidence: 99%

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen

1992

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“…The sensors and the relevant data acquisition module are optional. The versatility of the new apparatus was easily demonstrated [28,291. Note, however, that multicompartment electrophoresis instruments were not new, they had already been described by Theore11 [30] and Tiselius [31], two giants of classical biochemistry.…”

Section: The Rief Apparatusmentioning

confidence: 99%

Recycling isoelectric focusing and isotachophoresis

Bier

1998

Electrophoresis

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Of all electrophoretic methods, isoelectric focusing offers the highest resolution and is best suited for preparative applications. Over the years, several instruments were developed for this purpose, all operating in free fluids, in the absence of gels or other supporting matrices. In such systems, the avoidance of gravity or electrically driven convections is essential. Successful stratagems for fluid control included rapid recycling or rotation, in combination with either fine porosity screens or narrow gaps between parallel plates. The most successful apparatus so far is the Rotofor, in which fluid is stabilized by combining horizontal rotation with fine porosity screen partitioning. Recycling isotachophoresis offers the potential of separating proteins at high concentration. A new concept of tangential electrophoresis is described. To optimize the use of these devices for protein separation, low molecular weight, biologically acceptable buffers of known composition are essential. The buffering system developed for this purpose comprises a series of binary buffers that cover the pH range in steps of 1 pH unit or less. The pH gradient can be custom-designed and is of remarkable stability in operation.

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“…Good agreement was found for experimental values and those The second example presented here concerns the purification of a monoclonal antibody (mAb) from a mouse ascitic liquid. Some purifications were already reported, using liquid electrophoresis [25], IEF in carrier ampholytes or in immobilized pH gradients [26].…”

Section: Separation At Ph 93mentioning

confidence: 99%

Purification of bioproducts by free‐flow zone electrophoresis: Choice of processing parameters

1998

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Free-flow zone electrophoresis may be used to purify biological samples, due to differences in electrophoretic mobility, in absence of a matrix--most frequently a gel--thus enabling the biological integrity of even fragile molecules to be preserved. However, the process is more complicated than its principle suggests due to different transport phenomena interfering with electrophoretic migration, with the resultant separation depending both on separation effects and dispersive phenomena. The physical origin of the main effects involved was identified. Mathematical expressions were proposed to estimate the influence of the crescent effect and electrohydrodynamics on the process. In this paper, these equations are used to determine the minimum difference in electrophoretic mobility required for a separation to be achieved with respect to the processing parameters. A methodology is proposed which defines the conditions under which the difference in electrophoretic mobilities equals that calculated when considering the influence of dispersive phenomena. Optimized separations of the whey proteins lactoferrin and albumin, known to interact strongly, and the purification of a monoclonal antibody from a mouse ascitic fluid illustrate the approach.

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Rapid purification of antibodies to single band purity using recycling isoelectric focusing

Cited by 15 publications

References 10 publications

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen

Recycling isoelectric focusing and isotachophoresis

Purification of bioproducts by free‐flow zone electrophoresis: Choice of processing parameters

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