TMB 1-Component HRP MicroWell Substrate (SUBT)

Flow Cytometry, 1. Each 50 µL vial is sufficient for 1000 flow cytometry tests., 2. Spin thawed vial briefly in a microcentrifuge to remove any reagent that may have become trapped in the cap., 3. Dilute Green Live/Dead Stain concentrated stock solution (500 µM) 1:100 in PBS to prepare a 5000 nM working solution. For example, add 10 µL stock concentrate to 990 µL PBS. Prepare only what is needed for the experiment. Working solution should be used immediately, and any remaining diluted solution should be discarded., 4. Spike samples with a 1:100 dilution of the 5000 nM working solution. For example, spike 0.495 mL samples with 5 µL working solution., 5. Incubate samples for ~10 minutes at room temperature, protected from light., 6. Analyze with a flow cytometer using a blue laser at 488 nm and a 530/30 (FL1) emission filter setting, or similar., , Fluorescence Microscopy, 1. Each 50 µL vial is sufficient for 100 microtiter plate Wells., 2. Spin thawed vial briefly in a microcentrifuge to remove any reagent that may have become trapped in the cap., 3. Dilute Green Live/Dead Stain concentrated stock solution (500 µM) 1:100 in PBS to prepare a 5000 nM working solution. For example, add 10 µL stock concentrate to 990 µL PBS. Prepare only what is needed for the experiment. Working solution should be used immediately, and any remaining diluted solution should be discarded., 4. Spike samples with a 1:10 dilution of the 5000 nM working solution. For example, spike .45 mL samples with 50 µL working solution., 5. Incubate samples for ~10 minutes at room temperature, protected from light., 6. When bound to DNA, the peak absorption of Green Live/Dead Stain is 495 nm and the maximum emission is 512 nm. Visualize with a fluorescence microscope using optical filters that best approximate these settings.

TMB 1-Component HRP MicroWell Substrate (SUBT) is suitable for use in all ELISAs where the target detection level is in the ng-pg/mL range and horseradish peroxidase (HRP) is the conjugated detection enzyme. For assays requiring greater sensitivity, use TMB substrate SUBS, catalog no. 6275; for assays requiring less sensitivity, use TMB substrate SUBK, catalog no. 6277. SUBT should not be used for membrane or immunohistochemical applications. SUBT is a one-component, ready-to-use formulation containing 3,3’,5,5’-tetramethylbenzidine (TMB) in a mildly acidic buffer that does not contain aprotic solvents. TMB substrate is oxidized by the peroxidase enzyme to yield a soluble blue-green reaction product, which can be read at 370 nm or 620-650 nm. In endpoint assays, the reaction can be stopped by adding equal volumes of Stop Solution for TMB MicroWell Substrates (STOPT, catalog #6282). Addition of STOPT changes the chromagen color from blue-green to yellow, where it can be read at 450 nm, and concurrently stabilizes the yellow TMB product for one hour. Stopping the reaction will increase the sample absorbance value up to 3-fold. To avoid overdeveloping the TMB substrate reaction, the blue-green reaction product should be periodically monitored on an ELISA plate reader using 620-650 nm absorbance filter settings. When OD values reach approximately 0.7 units, the reaction should be stopped using STOPT. For best results, the absorbance should be monitored and read before values exceed 2.5 OD units. The substrate should not be diluted. The intensity of the reaction can be reduced by further dilution of the antibodies/conjugates used in the assay or by shortening the incubation time. TMB 1-Component HRP MicroWell Substrate is ready to use at 1X; add 100 µL to each Well. Best results are obtained by equilibrating the TMB substrate for one hour at room temperature (25°C) prior to use.