Submarine Gel Electrophoresis Purification of a Small Molecule:  99.9998 Fluorescently Pure IMI2

Abstract

HPLC and related techniques failed to raise the fluorescence purity of a BODIPY-imidazole dye (IMI2) above 99.5%, based on assessment of dye purity by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This difficulty was overcome by employing submarine gel electrophoresis, which raised the fluorescence purity level to about 99.9998%. This estimate of final dye purity was made by using immobilized metal affinity chromatography to selectively remove the dye from a sample that was then analyzed by CE-LIF as a 5000× more concentrated sample than the original. The amount of dye purified on a 87-× 67-× 10-mm (l × w × h) gel in a 40-min run was at the 0.5-mg level. The dye was recovered by soaking the sliced-out electrophoretic band in methanol. We are now in a position to employ this dye for labeling of trace phosphomonoester analytes without being hampered by dye impurities, by further developing methodology that we reported previously (Wang, P.; Giese, R. W. Anal. Chem. 1993, 65, 3518-3520). More generally, we conclude that submarine gel electrophoresis can be a convenient and useful separation technique for purifying small molecules.

Xanthamide Fluorescent Dyes

“…We have been exploring functionalized BODIPY dyes as tags for labeling and detection of DNA adducts, , but the limited stability of these dyes has impeded our work. , They are also quite expensive. Accordingly, we have sought an alternative dye.…”

Proteinase assay by capillary electrophoresis employing fluorescence-quenched protein-dye conjugates

“…Giese and co-workers utilized CE-LIF to assess the purity of a BOD-IPY-imidazole dye prior to purification by submarine gel electrophoresis [21] and to study the covalent bonding of a BODIPY-based dye to a phosphomonoester [22]. Giese and co-workers utilized CE-LIF to assess the purity of a BOD-IPY-imidazole dye prior to purification by submarine gel electrophoresis [21] and to study the covalent bonding of a BODIPY-based dye to a phosphomonoester [22].…”

Matrix‐assisted laser desorption/ionization mass spectrometry of deoxynucleotides labeled with an IMI dye

“…To overcome these problems, we are developing a new method, 'IMI-postlabeling,' for detecting DNA adducts. [3][4][5][6][7] The concept for this technique is the same as that of 32 Ppostlabeling, except that: (1) the labeling reagent is a fluorescent IMI dye (so called because imidazole is the functional group for labeling); (2) the labeling reaction is conducted chemically rather than enzymatically (a carbodiimide is used to activate the phosphomonoester of the DNA adduct for coupling to the IMI dye); (3) capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is used to detect the IMI-labeled DNA adducts; and (4), as demonstrated here, IMI-labeling facilitates detection of DNA adducts by MALDI-MS. 4 form by treatment with NH 4 OH to pH 9.6, followed by washing with water. IMI2 was prepared as a methanolic stock solution as described.…”

“…IMI2 was prepared as a methanolic stock solution as described. 7 2', 4', 6'-Trihydroxyacetophenone (THAP) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) were from Aldrich (Milwaukee, WI, USA).…”