Streptavidin-HRP

HSV in human fibroblasts was detected using biotin-labeled HSV DNA probes, streptavidin-HRP complex, and enhanced CL substrate reagent for HRP [56], The presence of HSV DNA was observed in cells infected with clinical samples known to contain the HSV virus fixed at 48 h postinfection, with a sharp topographical localization and a good preservation of cellular morphology. 

A CL ISH assay for simultaneous detection of two different viral DNAs (HSV and CMV DNAs) was developed utilizing both HRP and AP as reporter enzymes [63], A biotinylated HSV DNA probe and a digoxigenin-labeled CMV DNA probe were cohybridized with samples then CL detection of the two probes was performed. The HSV DNA was revealed using a streptavidin-HRP complex... 

Incubate with streptavidin-HRP conjugate and anti-digoxigenin antibody conjugate for 15-30 min at RT. Wash again thoroughly. Stain as described in Protocols 2.5.4 or 2.5.5. 

Incubate with the corresponding enzyme conjugate (e.g., anti-digoxigenin AP conjugate or streptavidin-HRP) for 30 min, wash carefully and perform the staining as described in Protocols 2.5.5 and 2.5.4. 

Protocol for the Preparation of Avidin—HRP or Streptavidin—HRP by Reductive Amination... 

Incubation is carried out with 100 xl primary antibody in a humid chamber for 30 min at room temperature. After being rinsed three times in TBS, the slides are incubated with rabbit antimouse antibody (1 200) in TBS for 30 min at the same temperature. They are rinsed three times in TBS and incubated with streptavidin/HRP P397 (DAKO) for 30 min at room temperature. Following rinsing three times in TBS, the slides are incubated for 4 min in DAB chromogen solution. 

Streptavidin HRP use in accordance with the manufacturers instructions. Numerous companies sell Streptavidin-HRP, including Sigma, Becton Dickinson, and Biosource. 

Make dilutions of streptavidin-HRP conjugate between 1 1000 and 1 40,000 and use to determine the optimum concentration for this reagent. 

In Far-Western blotting the membrane is probed with another protein to detect specific protein-protein interactions (24). The reaction can be revealed using biotinylated or GST-tagged bait or probe protein followed by a streptavidin-HRP or an anti-GST-HRP chemiluminescent detection system, respectively. 

Dilute streptavidin-HRP according to manufacturers instructions. Add 100 pL per well. Incubate at room temperature for 30 min. 

For smaller laboratories, the work involved in validation is often difficult, but there are two alternatives. Users can choose a system with an existing standardized and validated protocol and validated interpretation system. Commercially available kits generally provide these, and when utilized exactly as described in the kit insert, are guaranteed to provide diagnostically useful results. A second option would be to use one of the more common standard systems of fixatives with known antibodies, in which publication data has provided some evidence of functionality. As an example, a laboratory could use a 10% neutral buffered formalin fixation with a standard protocol, followed by a biotin-streptavidin HRP system, using a monoclonal antibody combination called AE1/AE3. This has been proven to be a reliable measure of cytokeratin in tissue sections. 

Peroxidase Block + Secondary Antibody + Streptavidin-HRP + DAB/AEC + Counterstain... 

Add biotinylated monoclonal antibody-streptavidin-HRP, Incubate 30min, 20°C. 

Figure 3.3.2 Format of sandwich ELISA for antigens (high molecular weight compounds). This format needs a capture antibody (on the microtitre plate surface) and a detector antibody, to which an enzyme (e.g. HRP) can be conjugated. Other possibilities would be that the detector antibody is labeled with a fluorophore or is biotinylated. The latter would use streptavidin-HRP in the detection step...

Fig. 6. (A) Caspase-7 forms generated by caspase cleavage Pro, pro-region LSU, large subunit and SSU, small subunit. (B) Western blot of cell extracts. Extracts from Jurkat cells incubated with STS for the indicated times and then for 1 h with bzVKD-fmk and STS were subjected to Western blotting with anti-caspase-7 LSU (Anti-LSU), anti-caspase-7 SSU (Anti-SSU), or HRP-streptavidin (HRP-S A). (C) Western blots of captured polypeptides. After incubation with STS for the indicated times and then 1 h with bzVKD-fmk and STS cells were extracted as described in Methods for capture on 6-well dishes coated with caspase-7 capture antibody. Captured material was solubilized in SDS sample buffer and subjected to Western blotting with anti-caspase-7 LSU (Anti-casp-7 LSU), anti-caspase-7 SSU (Anti-casp-7 SSU), or HRP-streptavidin (HRP-SA).

Caspase-7 LSU was the major captured bzVKD-fmk modified polypeptide detected by HRP-streptavidin (Fig. 6C). The amount of LSU detected at 1 h in shorter exposures (results not shown) of the anti-LSU blot was less than the amount detected at 2 1 h. Minor amounts of LSU-SSU and Pro-LSU covalently modified with bzVKD-fmk were present in captured material. Signal generated in the active caspase-7 ELISA is, therefore, primarily due to bzVKD-fmk modified LSU with minor contribution by bzVKD-fmk-modified LSU-SSU and Pro-LSU. All bzVKD-fmk containing bands had corresponding bands when blotted with anti-caspase-7 LSU except for one. Pro-LSU was detected in cell extracts (Anti-LSU blot, Fig. 6B) but was not readily detected by anti-LSU in captured material (Anti-LSU blot, Fig. 6C) whereas a band with the appropriate mobility was detected by HRP-streptavidin (HRP-SA blot, Fig. 6C). Although the basis for this discrep-... 

---