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Continuous Mode of Operation for Large Volume Dosing in Analytical Carrier Ampholyte-Free Isoelectric Focusing of Proteins Applied to Off-Line Detection of Fractions

Continuous Mode of Operation for Large Volume Dosing in Analytical Carrier Ampholyte-Free Isoelectric Focusing of Proteins Applied to Off-Line Detection of Fractions

Jana Budilová, Jirí Pazourek, Pavel Krásenský, Jan Pospíchal

J Sep Sci. 2006 Jul;29(11):1613-21.

PMID: 16922278

Abstract:

Mass spectrometry is being increasingly used for analysis of proteome complex samples. Sample preparation is often necessary to remove matrix interferences and to concentrate analytes prior to MS measurement. A useful method for this purpose is Carrier Ampholyte Free-Isoelectric Focusing (CAF-IEF). In this paper CAF-IEF of ampholytes was performed on a commercial apparatus EA101 (Villa Labeco, Slovakia) equipped with a specially made column for samples of large volume (up to 0.5 mL). A new continuous mode without voltage interruption or electrolyte replacement was developed. In this mode, a low molecular mass pI marker (PIM 7.4) and low concentrations of myoglobin and insulin (16 mg/L), respectively, were concentrated, and then 5-microL fractions collected for off-line analyses. The total time of focusing was 66 minutes. The concentration of PIM 7.4 in the fractions was increased up to 75 times (determined by UV-VIS spectrometry). The concentration in the fractions was increased up to 30 times for myoglobin and 10 times for insulin.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411148	UV-VIS detectable IEF Marker			Price

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