IMAC: Nonlinear elution chromatography of proteins

Vunnum, Suresh; Cramer, Steven M.

doi:10.1002/(sici)1097-0290(19970520)54:4<373::aid-bit11>3.0.co;2-h

Cited by 9 publications

(4 citation statements)

References 22 publications

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“…With 15-μL injections, the myoglobin peak was still eluting in the IgG front, but a second peak was observed at 1.7 min shortly after the injection peak, which was shown to contain myoglobin upon reanalysis of the corresponding fraction. Injections of 20 μL finally resulted in a significant increase in the signal intensity of the additional peak, which is probably a consequence of peak deformation and peak splitting due to overloading and nonlinear elution effects . We concluded that the maximum loading capacity of the column lies between 10 and 15 μL serum.…”

Section: Resultsmentioning

confidence: 81%

“…Injections of 20 µL finally resulted in a significant increase in the signal intensity of the additional peak, which is probably a consequence of peak deformation and peak splitting due to overloading and nonlinear elution effects. 36 We concluded that the maximum loading capacity of the column lies between 10 and 15 µL serum. Upon connection of an additional 50 mm × 4 mm i.d.…”

Section: Depletion Of High-abundant Serum Proteins Using Strongmentioning

confidence: 89%

See 1 more Smart Citation

Absolute Myoglobin Quantitation in Serum by Combining Two-Dimensional Liquid Chromatography−Electrospray Ionization Mass Spectrometry and Novel Data Analysis Algorithms

et al. 2005

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To measure myoglobin, a marker for myocardial infarction, directly in human serum, two-dimensional liquid chromatography in combination with electrospray ionization mass spectrometry was applied as an analytical method. High-abundant serum proteins were depleted by strong anion-exchange chromatography. The myoglobin fraction was digested and injected onto a 60 mm x 0.2 mm i.d. monolithic capillary column for quantitation of selected peptides upon mass spectrometric detection. The addition of known amounts of myoglobin to the serum sample was utilized for calibration, and horse myoglobin was added as an internal standard to improve reproducibility. Calibration graphs were linear and facilitated the reproducible and accurate determination of the myoglobin amount present in serum. Manual data evaluation using integrated peak areas and an automated multistage algorithm fitting two-dimensional models of peptide elution profiles and isotope patterns to the mass spectrometric raw data were compared. When the automated method was applied, a myoglobin concentration of 460 pg/microL serum was determined with a maximum relative deviation from the theoretical value of 10.1% and a maximum relative standard deviation of 13.4%.

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Section: Resultsmentioning

confidence: 81%

Section: Depletion Of High-abundant Serum Proteins Using Strongmentioning

confidence: 89%

Absolute Myoglobin Quantitation in Serum by Combining Two-Dimensional Liquid Chromatography−Electrospray Ionization Mass Spectrometry and Novel Data Analysis Algorithms

et al. 2005

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“…The technology is based on the biospecific association of molecules such as antibody−antigen, enzyme−inhibitor, enzyme−coenzyme/biomimetic dyes, and glycoprotein − lectin. During the past three decades, affinity chromatography has been extensively studied and applied in the downstream processing of proteins (1 − 3). At the same time, alternative affinity purification techniques have been developed, including affinity precipitation (4, 5), affinity cross‐flow filtration/ultrafiltration (6 − 8), affinity partitioning based on aqueous two‐phase extraction (9, 10), and affinity‐based reversed micellar extraction (11).…”

Section: Introductionmentioning

confidence: 99%

Protein Separation Using Affinity-Based Reversed Micelles

Sun

Tong

et al. 1999

Biotechnol. Prog.

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Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction. The affinity partitioning of lysozyme and bovine serum albumin (BSA) to the CB-lecithin micelles was studied. Formation of mixed micelles by additionally introducing a nonionic surfactant, Tween 85, to the CB-lecithin micelles was effective to increase the solubilization of lysozyme due to the increase of W0 (water/surfactant molar ratio)/micellar size. The partitioning isotherms of lysozyme to the CB-lecithin micelles with and without Tween 85 were expressed by the Langmuir equation. The dissociation constants in the Langmuir equation decreased on addition of Tween 85, indicating the increase of the effectiveness of lysozyme binding to the immobilized CB. On addition of 20 g/L Tween 85 to 50 g/L lecithin/hexane micellar phase containing 0.1 mmol/L CB, the extraction capacity for lysozyme could be increased by 42%. Moreover, the CB-lecithin micelles with or without Tween 85 showed significant size exclusion for BSA due to its high molecular weight. Thus, lysozyme and BSA were separated from artificial solutions containing the two proteins. In addition, the affinity-based reversed micellar phase containing Tween 85 was recycled three times for lysozyme purification from crude egg-white solutions. Lysozyme purity increased by 16-18-fold, reaching 60-70% in the recycled use.

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“…Martin del Valle and Galan [5] studied the kinetic and mass-transfer effects in affinity chromatography. Vunnum and Cramer [6] investigated how modulators influenced protein elution profiles in metal affinity chromatography. The simple scale-up rules, however, may be not accurate enough [1].…”

Section: Introductionmentioning

confidence: 99%