rev: February 23, 2001
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for Researchers and
READY TO USE TMB FOR BLOTTING
-see also TMB-SH for use in histochemistry
TMB FOR BLOTTING and DOT BLots
catalog# RDI-TMB-PPT $281.00/500ml $438.00/Liter $375.00/Liter 5+
USE: A single component precipitating Substrate for the localization of Horseradish Peroxidase Labeled Probes on western, Northern, Southern and Dot Blots ((histochemistry applications must use aqueous based reagents-no solvents).
approx 1.13mMol/L TMB. One component (premixed TMB and H202) . Stable for up to 18 months at room temp or up to 24 months at 2-8 DEG C.
Background:3,3'5,5'-Tetramethylbenzidine (TMB), when reacted with horseradish peroxidase (HRP) and hydrogen peroxide, forms a blue free radical cation 1-electron oxidation product. It is routinely used in ELISA procedures. For TMB to be worthwhile as a blotting regent, enhancers such as dextran sulfate are usually added. Lot-to-lot variations of molecular weights and degree of sulfonation of charged polysaccharides increase the complexity of manufacturing processes. This product has developed proprietary methodology that eliminates the use of sugar polymers and guarantees the production of a consistent single component precipitating TMB solution (TMB-PPT).
Method: After electropherograms have been transferred to appropriate membranes and reacted with HRP labeled probes, addition of TMB-PPT Solution results in the formation of aquamarine bands at the sites of HRP activity. Similarly, after addition of TMB-PPT Solution to suitable treated dot blots, aquamarine dots appear at the site of enzyme activity.
TMB-PPT SOLUTION: Contains TMB, 1.13 mMol L(-1), and hydrogen peroxide, 1.91 mMol L(-1) and less than 1% dimethylsulfoxide in a 0.08 Mol L(-1) acetate buffer, pH 4.9. Also contains non-toxic proprietary stabilizers.
Store at room temperature (15-28'C). Storage in refrigerator (2-8 'C) will not inhibit product performance. Warm to assay temperature prior to use.
Avoid exposure to direct sunlight.
Discard if solution becomes blue and turbid.
Sample PROCEDURE (must be optimized for each application).
1. After the final binding reaction with a HRP labeled probe, wash membranes thoroughly with phosphate buffered saline or tris buffered saline containing 0.1% Tween-20.
NOTE: Do not use reagents containing thimerosal or phenolic compounds as preservatives. Strange results will be ob- served if these compounds are present.
2. Following the final wash, cover the membranes with TMB-PPT Solution and incubate with gentle rocking for 5-30 minutes. Longer incubations may be necessary depending upon enzyme activity.
NOTE: If the color develops immediately, the TMB precipitate may flake off the membrane. If this occurs, further dilution of the HRP probe is recommended. A fine blue line circum- scribing the band or dot also suggesstd dilution of enzyme probe to be necessary.
3. Stop reaction by washing thoroughly in reagent grade water.
DO NOT WASH WITH BUFFER SOLUTIONS OR TAP WATER. These will cause fading. DO NOT USE ACIDS TO STOP THE REACTION. Acids will turn the bands yellow. Excessive background staining in- dicates incomplete removal of non-bound HRP from membranes. Increase the wash steps or washing time to remedy this problem.
4. Store dried membranes protected from light.
Aquamarine bands or dots will appear at the site(s) of enzyme activity.
For In Vitro or Further Manufacturing Use Only
For In Vitro Research or Further Manufacturing Use Only
RDI Divison of researchd Industries Intl
San Jose, 95123 CA Snell ave 658
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