Horseradish Peroxidase Substrates: FEMYTOGLOW TM Available sizes 25mL, 5mL 1mL 2L to 1L Product # Description of the Product Applications SHRPE111 Single bottle HRP Substrate & stable for 45 Days Immunoassays, Blotting SHRPM112 Single bottle HRP Substrate & stable for 45 Days Immunoassays, Blotting SHRPE213 Two bottle HRP Substrate & stable for 45 Days after mixing Immunoassays, Blotting SHRPM214 Two bottle HRP Substrates Immunoassays, Blotting SHRPMI215 Two bottle HRP Substrates Plus Immunoassays, Blotting Chemiluminescent Substrate for HRP Enzyme (Single Bottle) DIRECTIONS 1. Store the HRP reagent bottle, at -2 o C. 2. When you use the HRP substrate keep out the bottle and keep the bottle at 4-8 Cfor at least 12 hours. Take out the required amount in a clean container and equilibrate at room temperature for 3 minutes before use. 3. This reagent can be used for 45-6 days if stored properly in a tight cap container at 4-8 o C. 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.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 3 minutes after contacting substrate with HRP enzyme. Instruction for Blotting with Horse Radish Peroxide Substrates Streptavidin-HRP Conjugates 2. Transfer proteins to the membrane. 3. Block the membrane with blocking buffer (check blocking buffer with HRP substrate for background). 4. Incubate the membrane with primary antibody for 3 to 45 minutes. 5. Wash the membrane with washing buffer. 6. Incubate the membrane with biotin labeled secondary antibody for 3 to 45 minutes. 7. Wash the membrane with washing buffer. 8. Incubate the membrane with Streptavidin-HRP conjugate (diluted in MDLLC enzyme diluent or other source) 3 to 45 minutes. 9. Wash the membrane with washing buffer (MDLLC wash buffer or other source) 5 to 6 times. 1. Incubate the membrane with enzyme substrate for 2 to 3 minutes. 11. Drain the excess substrate from the membrane. 12. Wrap the membrane in a plastic. 13. Expose the membrane to film or take a picture with CCD camera. Secondary Antibody-HRP Conjugate 6. Transfer proteins to the membrane.
7. Block the membrane with blocking buffer. 8. Incubate the membrane with primary antibody for 3 to 45 minutes. 9. Wash the membrane with washing buffer. 6. Incubate the membrane with HRP labeled secondary antibody for 3 to 45 minutes. 13. Wash the membrane with washing buffer. 14. Wash the membrane with washing buffer (MDLLC wash buffer or other source) 5 to 6 times. 15. Incubate the membrane with enzyme substrate for 2 to 3 minutes. 16. Drain the excess substrate from the membrane. 17. Wrap the membrane in a plastic. 18. Expose the membrane to film or take a picture with CCD camera. Note: If you have any questions when you are using our HRP enzyme substrate, please contact us at the above address. Control No.: IMDLLC-7, Rev. 2. Chemiluminescent Substrate for HRP Enzyme (Two Components) DIRECTIONS 1. Store the reagent bottles, HRP-A and HRP-B, at 4-8 o C. 2. Mix equal volumes of HRP-A and HRP-B in a clean container and equilibrate at room temperature for 3 minutes before use. 3. Mixed reagent can be used for 45-6 days if stored properly in a tight cap container at 4-8 o C. 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 then gram depending on the source of the HRP enzyme. 6. For good results, wash your tube, microtiter plate or membrane with.2m phosphate buffer, ph 8.4 to 8.6, and use the substrate. 7. Best results for chemiluminescence can be obtained from 6 minutes to 3 minutes after contacting substrate with HRP enzyme. Instruction for Blotting with Horse Radish Peroxide Substrates Streptavidin-HRP conjugate 2. Transfer proteins to the membrane. 3. Block the membrane with blocking buffer (check blocking buffer with HRP substrate for background). 4. Incubate the membrane with primary antibody for 3 to 45 minutes. 5. Wash the membrane with washing buffer. 6. Incubate the membrane with biotin labeled secondary antibody for 3 to 45 minutes. 7. Wash the membrane with washing buffer. 8. Incubate the membrane with Streptavidin-HRP conjugate (diluted in MDLLC enzyme diluent or other source) 3 to 45 minutes. 9. Wash the membrane with washing buffer (MDLLC wash buffer or other source) 5 to 6 times. 1. Incubate the membrane with enzyme substrate for 2 to 3 minutes. 11. Drain the excess substrate from the membrane. 12. Wrap the membrane in a plastic. 13. Expose the membrane to film or take a picture with CCD camera. Secondary Antibody-HRP Conjugate 2. Transfer proteins to the membrane. 3. Block the membrane with blocking buffer. 4. Incubate the membrane with primary antibody for 3 to 45 minutes. 5. Wash the membrane with washing buffer. 6. Incubate the membrane with AP labeled secondary antibody for 3 to 45 minutes.
8. Wash the membrane with washing buffer. 9. Wash the membrane with washing buffer (MDLLC wash buffer or other source) 5 to 6 times. 1. Incubate the membrane with enzyme substrate for 2 to 3 minutes. 11. Drain the excess substrate from the membrane. 12. Wrap the membrane in a plastic. 13. Expose the membrane to film or take a picture with CCD camera. Note: If you have any questions when you are using our HRP enzyme substrate, please contact us at the above address. Control No.: IMDLLC-6, Rev. 2. Chemiluminescent Substrates for Horseradish Peroxidase Enzyme 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. The purity of horseradish peroxidase is related to the RZ (Reinheits Zahl) number which is the ratio (3.) of the absorbance at 43 nm and 275 nm. Pure horseradish peroxidase with an RZ of 3. contain several isozymes, the major component (isozymes C) has the highest activity and an RZ of 3.5 and can be isolated easily. 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 3 to 45 days after mixing at 4 o C. The stabilized formulations for the detection of horseradish peroxidase are covered by US Patent 6,62,679 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: 35 Sensitivity of Substrate 3 Intensity [RLU] 25 2 15 1 5 HRP. fg HRP 1.75 fg HRP 3.5 fg HRP 7. fg HRP 14. fg Concentration of HRP Enzyme
Sensitivity of Substrates Intensity [ RLU] 7 6 5 4 3 2 1 HRP 28. fg HRP 56. fg HRP 112. fg HRP 224. fg Concentration of HRP Enzyme Sensitivity of Substrate 6 5 Intensity [RLU] 4 3 2 1 HRP 224. fg HRP 448. fg HRP 896. fg HRP 1792. fg Concentration of HRP Enzyme
Intensity [ RLU] 2 18 16 14 12 1 8 6 4 2 Effect Of Temprature On Substrate HRP Subs tr ate Com ponents A and B at 37 degrees C for 3 days HRP Subs tr ate Com ponents A and B at 4 degrees C for 3 days 1 2 3 4 5 6 7 8 9 1 Time in fiv e minutes Intensity [RLU] 16 14 12 1 8 6 4 2 Substrate After 1 Days of Mixing Substrate After 1 days at 4 degree C Substrate Freash 1 2 3 4 5 6 7 8 9 Time in five minutes
Substrate after 63 days of Mixing Intens ity [RLU] 12 1 8 6 4 2 Substrate After 63 days at 4 degree C Substrate Freash 1 2 3 4 5 6 7 8 9 1 Tim e in five m inutes COMPARISON OF HORSERADISH PEROXIDASE ENZYME SUBSTRATE OF MDLLC AND THE LEADING COMPETITOR Intensity[ RLU] 9 8 7 6 5 4 3 2 1 COMPETITOR MDLLC 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Time in 5 minute