| 1. |
Store the reagent bottles at 2-6˚C or –20˚C. |
| 2. |
Equilibrate the reagent at room temperature till clear solution observed if stored at –20˚C. |
| 3. |
Do not contaminate the ß-Glucuronidase substrate with ß-Glucuronidase enzyme or other proteins. |
| 4. |
ß-Glucuronidase substrate has a wide range to detect ß-Glucuronidase enzyme in solution as well as on membrane.
The lower enzyme concentration may be atto grams and the highest concentration ß-Glucuronidase enzyme may be
nano gram depending on the source of the ß-Glucuronidase enzyme. |
| 5. |
To obtained good results, wash the tube or microtiter plate or membrane with 0.1M phosphate buffer, pH 7.2 to 7.4, and then use the substrate. |
| 6. |
Best results for chemiluminescence detection of ß-Glucuronidase enzyme can be obtained from 30 minutes to 45 minutes incubation of
ß-Glucuronidase substrate with ß-Glucuronidase enzyme and read the plate or tube within 2 to 10 minutes after triggering the reaction
mixture by accelerator or enhancer. |
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| Detection of ß-Glucuronidase in Microtiter Plate Luminometer |
| 1. |
Add 5 to 10ml of diluted ß-Glucuronidase enzyme or cell extract to microplate wells. |
| 2. |
Add 50 to 100ml of ß-Glucuronidase substrate and incubate for 30 to 60 minutes at room temperature. |
| 3. |
Add 50 to 100ml of triggering reagent and shake it for 10sec. |
| 4. |
Place the microtiter plate in luminometer and start reading. |
| 5. |
Best results are obtained between 2 to 10 minutes. |
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| Control No.: IMDLLC-08, Rev. 0 |
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| Chemiluminescent Substrates for ß-Glucuronidase Enzyme |
ß-Glucuronidase Enzyme is present in plant and mammalian cells. The E. coli GUS gene, which encodes
ß-Glucuronidase, is a major marker for detecting transformed plant cells. ß-Glucuronidase is widely used reporter genes in plant genetic research. Reporter genes are those that encode proteins that cab be unambiguously assayed once they are incorporated within a living cell. When reporter genes are fused with other genes or with genomic regulatory elements, the resulting DNA constructs can be introduced into the cell of interest, and the reporter gene product (an enzyme) can than be assayed. This technique can be used to identify DNA sequences or regulatory proteins that are required for proper gene expression. |
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| Our new series of ultra-sensitive 1, 2-dioxetanes can detect ß-Glucuronidase enzyme at very low level. MDLLC chemiluminescent system is at least 1000 times more sensitive compared to the chromophoric substrates. These novel 1, 2-dioxetanes are protected by US Patent 6,461,876B1and other pending PCT and US patent applications. |
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MDLLC substrates for ß-Glucuronidase enzyme are more sensitive due to the effect of electrons on the decomposition of 1,2-dioxetanes intermediate in the form of phenoxide ion formed after the interaction of glucuronic acid group on 1, 2-dioxetane and ß-Glucuronidase enzyme and can
be shown as: |
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