Rapid imaging, using a cooled charge‐coupled‐device, of fluorescent two‐dimensional polyacrylamide gels produced by labelling proteins in the first‐dimensional isoelectric focusing gel with the fluorophore 2‐methoxy‐2,4‐diphenyl‐3(2H)furanone

Abstract

A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…For labeling, 20 μg of protein dissolved in 0.01 M borate buffer (pH 9.5) was mixed with 60 μg of MDPF in dimethyl sulfoxide (DMSO). In an independent study, MDPF was also applied to IEF, 1D and 2D post-electrophoretic gel staining [138]. For post-IEF staining, gels were shaken with 0.02 moles of MDPF in MeOH/0.2 M sodium borate buffer (pH 9.5) and washed in MeOH and water prior to second-dimension separation [138].…”

“…In an independent study, MDPF was also applied to IEF, 1D and 2D post-electrophoretic gel staining [138]. For post-IEF staining, gels were shaken with 0.02 moles of MDPF in MeOH/0.2 M sodium borate buffer (pH 9.5) and washed in MeOH and water prior to second-dimension separation [138]. For post-SDS-PAGE staining proteins were fixed and stained with 3.8 mM MDPF for 1D gels and 0.95 mM MDPF with longer staining time for 2D gels [138].…”

“…For post-IEF staining, gels were shaken with 0.02 moles of MDPF in MeOH/0.2 M sodium borate buffer (pH 9.5) and washed in MeOH and water prior to second-dimension separation [138]. For post-SDS-PAGE staining proteins were fixed and stained with 3.8 mM MDPF for 1D gels and 0.95 mM MDPF with longer staining time for 2D gels [138]. An LDR between 50 and 300 ng (soluble human lymphoid cell line IM9 protein) was identified but this is clearly only a portion of that determined using the pre-electrophoretic MDPF method, and as such does not indicate a quantitative advantage over pre-labeling [138].…”

“…In 1988, the use of the CCD 2200 Imaging System which boasted 385× 578 pixels was reported [138]. The maximum signal detectable by this camera was 10 5 electrons while the read noise or minimum signal was 1 electron.…”

Protein stains for proteomic applications:Which, when, why?

“…Several dyes have been reported for fluorescence staining of proteins either before or after electrophoresis (reviewed in the introduction of [56]). Fluorescamine was utilized more than 25 years ago to stain 2-D patterns of nonhistone chromatin proteins [57].…”