Conjugated-Streptavidin horse radish peroxidase
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Peroxidase-conjugated Streptavidin
Streptavidin is a protein derived from Streptomyces avidinii. Streptavidin binds biotin (vitamin B7) in one of the strongest non-covalent interactions known.
The HorseRadish Peroxidase enzyme is made visible when it catalyzes oxidation of a substrate by hydrogen peroxide. Substrates are chosen which change color on oxidation, and the presence of the HRP or an HRP-conjugate can then be detected by spectrophotometry. In this case the HRP is conjugated to Avidin, which binds to the biotin on MAB211p, which is the monoclonal antibody binding specific epitopes on SeM.
The substrate used in the SeM ELISA protocol is tetramethylbenzidine (TMB). When oxidized by HRP it turns blue (read absorbance at 650 nm), but following stopping the reaction using 0.6 N HCL, it turns yellow (read absorbance at 450 nm)
| Add 1.0 mL dH2O to 1 vial (1.0 mg) freeze dried powder | ____ | (1 mg/ml) |
| Centrifuge product if not clear | ____ | |
| Store at 2-8 C | ____ | |
| Label vial "exp" followed by date 6 weeks in the future | ____ |
Should be done fresh each day:
| Dilution | 0.1 ug/mL | 1 ug/mL* | 2 ug/mL |
| Check dilution used | ____ | ____ | ____ |
| Final "Dilution" | 1:100,000 | 1:10,000* | 1:5,000 |
| Peroxidase-HRP stock diluted 1:10 (1 mg/mL) | 1 uL | 10 ul* | 20 ul |
| Volume of wash buffer (0.15M PBS, 0.05% tween 20) | 10 mL | 10 mL* | 10 mL |
*This is the dilution specified in the SeM ELISA Protocol.
HRP-Streptavidin provides more technical details.
Ordering Information:
www.jacksonimmuno.com
Jackson Immunoresearch, Peroxidase-conjugated streptavidin, 1.0 mg as freeze-dried powder. The catalog # is 016-030-084.
TMB provides more detailed technical information
Ordering Information:
www.surmodics.comTMB One Component HRP Microwell Substrate # 1000-01 TMBW 100mL