FACEÂ® N-Linked Oligosaccharide Profiling Kit - ProZyme

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OLIGO Quantitation Control (E5) The OLIGO Quantitation Control (E5) consists of 6 nmoles of unlabeled maltotetraose (Glucose 4 ). Once this control is reconstituted, 10 µl of E5 will contain 200 pmoles of maltotetraose. E5 may be used as an internal labeling control, provided that you verify that your sample does not contain an oligosaccharide that will co-migrate with maltotetraose (e.g. Man 3 GlcNAc 2 migrates very close to Glucose 4 ). As shown in Figure 3 (page 24), the labeling efficiency of your sample is greater than 95% when labeling less than 20 nmoles. With some unknown samples you may not know how much sugar you are labeling before you set up the digest. E5 is provided so that a "spike" of 200 pmoles (10 µl) may be added to the enzyme digest prior to the ethanol extraction step so that you can monitor labeling efficiency. The maltotetraose band from the "spike" will be clearly visible on the gel (it will comigrate with the Glucose 4 band in the OLIGO Ladder Standard (E3) as long as you load greater than 1/40 th of the labeling reaction in the lane. After correcting for dilution of the labeling reactions you will be able to determine the labeling efficiency of your sample by comparing the quantity of the internal Glucose 4 band against the same band in a lane containing (E5) labeled in a separate tube, or the Glucose 4 band in the lane containing the OLIGO Ladder Standard (E3). If you find that the quantity of your spiked internal Glucose 4 band (corrected for dilution) is only 190 pmoles (100% labeling efficiency would give you 200 pmoles), the labeling efficiency of the sample is about 95%. Page 7
Summary of Profiling Protocol Purified Glycoprotein N-Glycanase Digestion 2 hours to overnight Free Oligosaccharides Label with fluorophore 3 hours to overnight Labeled Oligosaccharides Load onto FACE Gel 30 minutes Electrophoresis 1 - 1 ½ hours FACE Imaging and Data Analysis Page 8
Page 1 and 2: FACE ® N-Linked Oligosaccharide Pr
Page 4 and 5: FACE N-Linked Oligosaccharide Profi
Page 6 and 7: Additional Reagents and Equipment R
Page 8 and 9: Description of the FACE N-linked OL
Page 12 and 13: Protocols SECTION 1 ENZYMATIC RELEA
Page 14 and 15: SECTION 2 LABELING OF OLIGOSACCHARI
Page 16 and 17: 3a Load 4 µl directly onto the gel
Page 18 and 19: 2 Pour pre-cooled OLIGO Gel Running
Page 20 and 21: 14 When the electrophoresis is comp
Page 22 and 23: 2 Develop film according to manufac
Page 24 and 25: TROUBLESHOOTING GUIDE Sample not mo
Page 26 and 27: Separation of N-Linked Oligosacchar
Page 28 and 29: 1 2 Figure 4. Lane 1 E3 - FACE Oli

FACEÂ® N-Linked Oligosaccharide Profiling Kit - ProZyme

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