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11758888001 Roche

HNPP Fluorescent Detection Set

Synonym: fluorescent detection set



Related Categories Fluorescent, Molecular Biology, Nucleic Acid Detection Substrates and Reagents, Nucleic Acid Detection and Hybridization
Quality Level   100
assay   >90% (Fast Red TR)
  >98% (HNPP, HPLC)
mol wt   Mr 257.2
  Mr 419.37 (HMPP)
mfr. no.   Roche
λmax   290 nm
shipped in   wet ice
storage temp.   2-8°C


General description

HNPP is a fluorescent substrate for alkaline phosphatase that enables sensitive detection of biomolecules. Upon dephosphorylation, HNPP produces a strongly fluorescent precipitate on membranes with a maximum of absorbance at 290nm and a maximum of fluorescence emission at 509nm.
The dephosphorylated form of HNPP, HNP, alone possesses weak affinity to tissues and chromosome preparations. For in situ applications HNPP is coupled with the diazonium salt Fast Red TR. The reaction product generated after dephosphorylation (HNP/Fast Red TR) is a highly fluorescent precipitate that accumulates during the reaction. The accumulation can be triggered by repeated addition of fresh substrate solution. Therefore, the HNPP-azo dye method enables enhanced detection sensitivity compared to conventional non-enzymatic fluorescence in situ detection methods employing fluorescein- or rhodamine-labeled antibody or avidin.


In combination with Fast Red, HNPP Fluorescent Detection Set is used for fluorescent detection of a nonradioactively labeled nucleic acid probe in in situ hybridization. HNPP/Fast Red gives a bright, long-lasting red or orange signal withstanding long exposures to UV light. HNPP alone can also be used for membrane blotting applications in which it gives a precipitating distinct signal. It is specifically suited for highly sensitive detection of nonradioactively labeled nucleic acids in a variety of applications:
• Fluorescence in situ hybridizations (FISH)
• Southern blots
• Northern blots
• Colony or plaque hybridizations
• Nonradioactive DNA sequencing blots
• Immunofluorescence
• Detection of alkaline phosphate-conjugated antibodies and alkaline phosphate activity
The sensitivity of detection of nonradioactive Southern blots is equivalent to NBT/BCIP and CSPD, after a 2-hour to overnight incubation. Single-copy sequences down to 1kb in length can be detected on metaphase chromosomes.


1 set containing 2 components.

Preparation Note

Working solution: Preparation of HNPP/Fast Red TR mix
Mix the following components in a sterile tube:
10 μl HNPP (10 mg/ml in dimethylformamide)
10 μl Fast Red TR solution.
Note: Do not use the solution longer than 4 weeks as progressive precipitation occurs.

1 ml buffer 4 (detection buffer)
Pass the HNPP/Fast Red TR mix through a 0.2 μm nylon syringe filter before use.Note:
It is recommended to use the HNPP/Fast Red TR mix freshly prepared. It may be stored up to a few days at 2 to 8 °C protected from light. Always pass the HNPP/Fast Red TR mix through a 0.2 μm nylon syringe filter immediately before use.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit component only


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HNPP, in dimethylformamide (DMF) 10 mg/ml; 23.8 mM    
Fast Red TR, powder    
Safety & Documentation

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Protocols & Articles


HNPP Fluorescent Detection Set Troubleshooting

II. Crystal-like structures Storage of slide preparations When storing the stained slide preparations for several weeks, a crystal-like structure might be formed, coating tissues and chromosomes. Thu...
Keywords: Centrifugation, Nucleic acid hybridization, Precipitation, Reductions

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