Effect of separation dimensions on resolution and throughput using very narrow‐range IEF for 2‐DE after solution phase isoelectric fractionation of a complex proteome

Abstract

This paper describes how the lengths of the first and second dimensions in narrow pH-range 2-DE affect the number of detected protein spots, by analysis of human breast carcinoma cell line lysates prefractionated by solution phase IEF. The aim is to maximize throughput while minimizing experimental costs. In this study, systematic evaluation of narrow-range IPG strip lengths showed that separation distances were very important, with dramatic increases in resolution when longer gels were used. Compared with 7 cm minigels, maximal resolution was obtained using 18 and 24 cm IPG strips. Systematic evaluation of SDS-PAGE gel length showed a far weaker influence of separation length on resolution in the second dimension compared with that observed for the IEF dimension. There was little benefit in using separation distances greater than 12-15 cm, at least with currently available electrophoresis units. The work shows that regions of the IPG strip not containing any proteins can be excised to fit a smaller gel if prefractionation using IEF in solution has been performed. As expected, larger 2-DE gel volumes resulting from the use of longer IPG strips and second dimension gels decreased detection sensitivity when equal protein loads were used. However, this effect could be readily eliminated by increasing the loads applied to IPG strips.

Mono-PEGylation of Alpha-MMC and MAP30 from Momordica charantia L.: Production, Identification and Anti-Tumor Activity

“…The voltage was increased to 100 V for 15 min, to 200 V for 15 min, and to 300 V for 60 min. The gel was stained by Coomassie brilliant blue R250 and the pI was confirmed by the distance against Amersham calibration kits, pH 3.0–10.0, as standard [18,19].…”

A Novel Method for Simultaneous Production of Two Ribosome-Inactivating Proteins, α-MMC and MAP30, from Momordica charantia L

“…The voltage was increased with 100 v/15 min, 200 v/15 min and 450 v/60 min. Bands in the gels were stained by coomassie brilliant blue R-250 and confirmed pI was based on the distance against standard [21], [22].…”