A Method for Monitoring the Glycosylation of Recombinant Glycoproteins from Conditioned Medium, Using Fluorophore-Assisted Carbohydrate Electrophoresis

Friedman, Yali E.; Higgins, Elizabeth

doi:10.1006/abio.1995.1342

Cited by 26 publications

(10 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This fluorophore is compatible with SDS and enzymes such as PNGase F, neuraminidase, alkaline phosphatase, and exoglycosidases. ANTS-derivatized sialylated and neutral oligosaccharides are prepared under mild conditions resulting in no oligosaccharide decomposition, often affording a single band on PAGE (71). A recent review details this method of using fluorophore labeling to reduce carbohydrates in PAGE analysis (54).…”

Section: Page Analysis Of Derivatized Carbohydratesmentioning

confidence: 99%

Electrophoresis for the Analysis of Acidic Oligosaccharides

Koketsu

Linhardt²

2000

Analytical Biochemistry

View full text Add to dashboard Cite

Section: Page Analysis Of Derivatized Carbohydratesmentioning

confidence: 99%

Electrophoresis for the Analysis of Acidic Oligosaccharides

Koketsu

Linhardt²

2000

Analytical Biochemistry

View full text Add to dashboard Cite

“…Glycoproteins can be separated by 2-D gel electrophoresis. However, glycosylated proteins are difficult to detect using typical gel approaches [26,27]. The most widely used methods for the detection of glycoproteins are autoradiography after incorporation of 3 H or 14 C sugars into cultured cells or tissues, nonradioactive detection using a periodic acid Schiff base procedure, or a fluorescence-based detection system.…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Lenchik

Pabst

et al. 2005

Electrophoresis

View full text Add to dashboard Cite

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.

show abstract

“…Alkaline phosphatase removes phosphate groups from oligosaccharides containing mannose 6-phosphate residues (19). Figure 2B shows that alkaline phosphatase treatment completely eliminated the mannose 6-phosphate peak in the glycoprotein without any effect on the other contaminant peaks ( Fig.…”

Section: Separation Of Mannose 6-phosphate Released From Rh␣gal By Hpmentioning

confidence: 96%

Mannose 6-Phosphate Quantitation in Glycoproteins Using High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection

Zhou¹,

Kyazike²,

Edmunds³

et al. 2002

Analytical Biochemistry

View full text Add to dashboard Cite

A Method for Monitoring the Glycosylation of Recombinant Glycoproteins from Conditioned Medium, Using Fluorophore-Assisted Carbohydrate Electrophoresis

Cited by 26 publications

References 0 publications

Electrophoresis for the Analysis of Acidic Oligosaccharides

Electrophoresis for the Analysis of Acidic Oligosaccharides

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Mannose 6-Phosphate Quantitation in Glycoproteins Using High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection

Contact Info

Product

Resources

About