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HRP Oligonucleotide Probes

HRP In Situ Hybridization Probes

Non-radioactive Horseradish Peroxidase (HRP) oligonucleotide probes are highly sensitive diagnostic tools. HRP DNA probes have been used for in situ hybridization methods for the detection, enumeration, and localization of specific target sequences in whole fixed cells. Horseradish peroxidase is a 45 kDa large enzyme isolated from horseradish plants. It catalyzes the oxidation of many substrates in the presence of H2O2, by converting non-fluorescent reduced form to fluorescent oxidized forms. Studies have shown that by using a combination of enzyme HRP probes and the tyramide signal amplification system (TSA), also known as CARD-FISH, FISH signals are up to 20-fold brighter. (Schönhuber et al., 1997). This "Catalyzed Reporter Deposition - Fluorescent in situ hybridization" (CARD-FISH) method represents an excellent tool for quantitative detection of microorganisms.

Bio-Synthesis’ HRP FISH probes are manufactured under strict quality control using mass spectrometry, analytical HPLC and HRP enzymatic activity assay.

Benefit of HRP Oligonucleotide Probes

  • Enhanced Sensitivity
  • Flexible Assay design applies directly to labeled fluorophore probes or probes in combination with TSA system , anti-HRP antibodies
  • Applicable to fluorescence microscopy and flow cytometry
  • Ease of use - no special equipment needed

HRP Oligonucleotide Services

  • Oligonucleotides up to 25 bases
  • Purification: diafiltration, Gel permeation chromatography or Ion-exchange chromatography
  • QC by MS, analytical HPLC and HRP enzymatic activity assay
  • Highest enzymatic activity with maximal signal

Price: Standard price covers the labor and materials, including the cost of HRP and it's labeling, cost of maleimide-activated oligo preparation, conjugation procedure, conjugate purification, and analysis. Please contact us for a quotation.

Quantity/Yields:

Guaranteed Amount Purification Options
Semi Purification Extended Purification
1 OD Dialysis SEC and ion exchange chromatography
2 OD Dialysis SEC and ion exchange chromatography
4 OD Dialysis SEC and ion exchange chromatography
10 OD Dialysis SEC and ion exchange chromatography
Extended Purification and QC option also available upon request.

Chemistry:

Our Oligonucleotide HRP labeling is based on one step conjugation of 5'-modified oligonucleotide directly linked to several available HRP carboxylates via EDC coupling method. This method is superior to the use of a two-step process as provided by other vendors. There is no need to introduce bifunctional cross linker. This method results in greater than 90% coupling efficiency that affords a desired 1:1 oligonucleotide/protein conjugates. Final conjugate are further purified by HPLC chromatography. PAGE is not recommended as it impairs protein activity.

Biosynthesis Inc.

The above image demonstrates oligo conjugation purity and functional assay, showing the activity of enzyme after conjugation.

General Procedure

  • Thiolated oligonucleotide during oligonucleotide synthesis and purified with HPLC.
  • Prepare HRP by reacting with heterobifunctional crosslinkers (i.e. NHS-ester/maleimide compounds), dialysis to remove excess cross linkers
  • Conjugate of HRP-maleimide to HS-oligonucleotide
  • Purify conjugate from excess of the free HRP-maleimide intermediate as well as free HRP
  • Characterize final product

Applications of Oligo-HRP Probes

  • CARD / FISH
  • FISH
  • Cytology
  • Histology
  • Immunoblotting
  • Immunosorbent assays