14530 Sigma-Aldrich

bisBenzimide H 33258

for fluorescence, ≥98.0% (HPLC)

Synonym: 2′-(4-Hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi(1H-benzimidazole) trihydrochloride, 2-[2-(4-Hydroxyphenyl)-6-benzimidazoyl]-6-(1-methyl-4-piperazyl)benzimidazole trihydrochloride, BBIH, BXI-72, HOE 33258, Hoechst 33258, NSC334072

  • CAS Number 23491-45-4

  • Empirical Formula (Hill Notation) C25H24N6O · 3HCl

  • Molecular Weight 533.88

  •  Beilstein Registry Number 4088183

  •  EC Number 245-690-6

  •  MDL number MFCD00012679

  •  PubChem Substance ID 57647172



Related Categories Alphabetical, Biochemicals and Reagents, Detection, Fluorescent Probes, Labels, Particles and Stains, Fluorescent Stains,
grade   for fluorescence
assay   ≥98.0% (HPLC)
form   powder
solubility   water and ethanol: 10 mg/mL
  H2O: soluble
  ethanol: soluble
fluorescence   λex 338 nm; λem 505 nm (pH 7.0)
  λex 355 nm; λem 465 nm in TE buffer; DNA
storage temp.   2-8°C


Other Notes

Useful reagent for cytogenetic studies; for the fluorescent staining of DNA in cells; simple two-step fluorimetric assay of DNA in isolated nuclei, cell extracts, or crude homogenates using Hoechst 33258


A bibenzimidazole dye that has been utilized as a fluorescent stain. It is membrane-permeable and selectively binds to adenine-thymine regions in the minor groove of B-form DNA with a high binding constant, making it a useful stain for DNA, chromosomes, and nuclei . It has been shown that the fluorescence is relatively insensitive to the presence of various solution reagents (i.e., BSA, RNA, Tris, sucrose, TEA/EDTA, Triton X-100, and SDS). Furthermore, the solution ionic strength and pH has been shown to be independent of the Stokes’ shift of bisBenzimide H 33258 . The properties of this dye make it useful in several applications such as detecting mycoplasma contamination in tissue culture , indicating early onset of apoptosis, sorting living cells based on DNA content, and acting as a probe for DNA-Drug interactions. Utilized in the design and development of a fluorimetric assay to quantitate cell numbers in microtiter plates. The methodology of the assay is based on Hoechst 33258 binding to DNA and is sensitive for a thousand cells. One application of this is assay is the normalization of ELISA data. The illumination of Hoechst 33253 coupled with Merocyanine 540 can be utilized as an effective method to eradicate bacterial infections (i.e., A. laidlawii contaminated corneal endothelial cell cultures) . BisBenzimide H 33258 is commonly used in many established staining protocols (David M. Terrian, Cancer Cell Signaling – Methods and Protocols,
Springer, Volume 218 ).

bisBenzimide H 33258 is useful for staining DNA, chromosomes and nuclei. bisBenzimide H 33258 may be used for fluorescence microscopy or flow cytometry.
Excitation max. = 346 nm
Emission max. = 460 nm

Biochem/physiol Actions

bisBenzimide H 33258 is a potent and selective Bcl-XL inhibitor. bisBenzimide H 33258 is a membrane-permeable, fluorescent DNA stain with low cytotoxicity that intercalate in A-T regions of DNA.

Safety & Documentation

Safety Information

GHS07  GHS07
Signal word 
Hazard statements 
Precautionary statements 
Personal Protective Equipment 
NONH for all modes of transport
WGK Germany 

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