UMP/CMP-Glo™ Glycosyltransferase Assay
Universal Glycosyltransferase Activity Assay
- Easy, "add-incubate-read" protocol
- Detects sialyltransferases and phosphoglycosyltransferases that use CMP-, CDP- or UDP-sugars as donor substrates
- Ideal for low-activity glycosyltransferases
Catalog Number:
Size
Catalog Number: VA1130
Catalog Number: VA1131
Catalog Number: VA1132
A Simple, Bioluminescent Assay for Glycosyltransferase Activity
The UMP/CMP-Glo™ Glycosyltransferase Assay is a homogeneous, single-reagent-addition method to rapidly detect UMP or CMP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of UMP/CMP Detection Reagent is added to simultaneously convert the UMP or CMP product to ATP and generate light in a luciferase reaction. The light generated is detected using a luminometer, and the light output is proportional to the concentration of UMP or CMP from low nM to 50μM. Luminescence can be correlated to UMP or CMP concentration by using a UMP or CMP standard curve.
Add-Mix-Read for Results
Start with purified enzymes, sugar donors and acceptor molecules, and you can detect the reaction products UMP or CMP from phosphoglycosyltransferases or sialyltransferases, respectively. Just add the UMP/CMP Detection Reagent and measure luminescence.
Works with Many Sialyltransferases and Phosphoglycosyltransferases
The UMP/CMP-Glo™ Assay can be used to measure the activity of just about any sialyltransferase that uses CMP-sugars or any phosphoglycosyltransferase that uses UDP-sugars as a substrate. Here, we looked at sialyltransferases involved in protein O-glycosylation.
Ideal for Low-Activity Glycosyltransferases and Phosphoglycosyltransferases
The sensitivity of the UMP/CMP-Glo™ Glycosyltransferase Assay means it can detect low UMP or CMP concentrations with high dynamic range. Therefore, whether assaying for a low-activity glycosyltransferase whose sugar transfer rate is low or using a low concentration of enzyme that produces a small amount of UMP or CMP, you can obtain a high signal-to-background ratio using the assay.
The data below show the linearity and sensitivity of the UMP/CMP-Glo™ Glycosyltransferase Assay. Here, a UMP or CMP standard curve was prepared over the indicated concentration range in 25µl of 1X glycosyltransferase reaction buffer in a solid white 96-well plate. UMP/CMP-Glo™ Glycosyltransferase Assay was performed using 25µl of UMP/CMP Detection Reagent at room temperature. Luminescence was recorded using a GloMax® luminometer. Values represent the mean of two replicates. The tables show luminescence measured 1 hour after adding the UMP/ CMP Detection Reagent, and signal-to-background ratios calculated for each concentration of the standard curve.
Luminescent Signal is Stable Over Several Hours
The UMP/CMP-Glo™ Assay generates a highly stable luminescent signal that is stable for more than 3 hours, making it convenient to batch-process assay plates.
The data here show that the signal obtained with the UMP/CMP-Glo™ Assay is stable for several hours. To determine signal stability, luminescence was recorded at 1 and 3 hours after adding detection reagent. The signal-to-background ratio did not change over the time measured (data not shown). RLU = relative light units.
Protocols
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
ATP Detection Substrate |
V363C | 1 × 1 vial |
Nucleotide Detection Buffer |
V696A | 1 × 5ml |
UMP, 10mM |
VA113A | 1 × 100μl |
CMP, 10mM |
VA114A | 1 × 100μl |
UMP/CMP-Glo™ Enzyme |
VA115A | 1 × 200μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
See Protocol for detailed storage recommendations.
What's in the box?
| Item | Part # | Size |
|---|---|---|
|
ATP Detection Substrate |
V363A | 1 × 1 vial |
Nucleotide Detection Buffer |
V696B | 1 × 10ml |
UMP, 10mM |
VA113A | 1 × 100μl |
CMP, 10mM |
VA114A | 1 × 100μl |
UMP/CMP-Glo™ Enzyme |
VA115B | 1 × 400μl |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
See Protocol for detailed storage recommendations.
What's in the box?
| Item | Part # | Size |
|---|---|---|
|
ATP Detection Substrate |
V363B | 1 × 1 vial |
Nucleotide Detection Buffer |
V696C | 1 × 100ml |
UMP, 10mM |
VA113A | 1 × 100μl |
CMP, 10mM |
VA114A | 1 × 100μl |
UMP/CMP-Glo™ Enzyme |
VA115C | 4 × 1ml |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
See Protocol for detailed storage recommendations.
Resources
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Frequently Used With
Ultra Pure UDP-Sugar Substrates
Substrates for use with the UDP-Glo™ Glycosyltransferase Assay.
V7071, V7072, V7081, V7082, V7091, V7092, V7171, V7172, V7321, V7322
GloMax® Explorer System
Easy-to-use, flexible multimode detection system.
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GloMax® Discover System
High-performance microplate reader for detecting luminescence, fluorescence and absorbance.
GM3000
GloMax® Navigator System
Dedicated microplate luminometer.
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