| 1. |
Store the HRP reagent bottle, at -20oC. |
| 2. |
When you use the HRP substrate keep out the bottle and keep the bottle at 4-80Cfor at least 12 hours. Take out the required amount in a clean container and equilibrate at room temperature for 30 minutes before use. |
| 3. |
This reagent can be used for 45-60 days if stored properly in a tight cap container at 4-8oC. |
| 4. |
Do not contaminate the HRP enzyme substrate with HRP enzyme or other proteins. |
| 5. |
HRP enzyme substrate has a wide range to detect HRP enzyme in solution as well as on membrane. The lower enzyme concentration may be a few femto grams and the highest concentration HRP enzyme may be one nano gram depending on the source of the HRP enzyme. |
| 6. |
For good results, wash your tube, microtiter plate or membrane with 0.2M phosphate buffer, pH 8.4 to 8.6, and then use the substrate. |
| 7. |
Best results for chemiluminescence can be obtained from 6 minutes to 30 minutes after contacting substrate with HRP enzyme. |
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| Chemiluminescent Substrate for HRP Enzyme (Two Components) |
| DIRECTIONS |
| 1. |
Store the reagent bottles, HRP-A and HRP-B, at 4-8oC. |
| 2. |
Mix equal volumes of HRP-A and HRP-B in a clean container and equilibrate at room temperature for 30 minutes before use. |
| 3. |
Mixed reagent can be used for 45-60 days if stored properly in a tight cap container at 4-8oC. |
| 4. |
Do not contaminate the HRP enzyme substrate with HRP enzyme or other proteins. |
| 5. |
HRP enzyme substrate has a wide range to detect HRP enzyme in solution as well as on membrane. The lower enzyme concentration may be a few femto grams and the highest concentration HRP enzyme may be one nano gram depending on the source of the HRP enzyme. |
| 6. |
For good results, wash your tube, microtiter plate or membrane with 0.2M phosphate buffer, pH 8.4 to 8.6, and then use the substrate. |
| 7. |
Best results for chemiluminescence can be obtained from 6 minutes to 30 minutes after contacting substrate with HRP enzyme. |
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Chemiluminescent Substrates for Horseradish Peroxidase Enzyme |
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Horseradish peroxidase enzyme is the most widely used in EIA and EIH. Typical peroxidases are hemoproteins and transfer hydrogen from hydrogen donors to hydrogen peroxide. Horseradish peroxidases often occur as multiple isozymes and are widely distributed particularly in plants. Three main types of horseradish peroxidases have been identified (i) the acidic horseradish peroxidases with a very high carbohydrate content; (ii)horseradish peroxidases with a pI around neutrality with a somewhat lower sugar content; and (iii) very basic horseradish peroxidases (pI>11) of low sugar content. |
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The purity of horseradish peroxidase is related to the RZ (Reinheits Zahl) number which is the ratio (3.0) of the absorbance at 403 nm and 275 nm. Pure horseradish peroxidase with an RZ of 3.0 contain several isozymes, the major component (isozymes C) has the highest activity and an RZ of 3.50 and can be isolated easily. |
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Our stabilized ultra-sensitive Luminol based substrates can detect horseradish peroxidase at femtogram level. MDLLC chemiluminescent system is at least 3 to 5 times more sensitive compared to the best competitor. These reagents are stable for 30 to 45 days after mixing at 4oC. The stabilized formulations for the detection of horseradish peroxidase are covered by US Patent 6,602,679 and other pending PCT and US patent applications. The kinetic results of low level of horseradish peroxidase enzyme and MDLLC substrate can be shown as: |
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