Manual

Product Number: RPA054

Shipping and Storage

Store at -20°C or -80°C, valid for 1 year. Avoid repeated freeze-thaw cycles as much as possible. Ship on dry ice under low temperature.

Component

Description

Endonuclease IV, also known as Nfo, is a multifunctional endonuclease involved in DNA damage repair. Its primary function is to recognize apurinic/apyrimidinic (AP) sites in double-stranded DNA molecules, and cleave the first phosphodiester bond at the 5′ side of the AP site to generate 3′-hydroxyl and 5′-deoxyribose phosphate (dRP) termini (Figure 1). In addition, this enzyme exhibits 3′-diesterase activity, which can release phosphoglyceraldehyde, intact deoxyribose 5′-phosphate and phosphate from the 3′ end of DNA; it also possesses 3′-5′ exonuclease activity to remove nucleotides from the DNA strand in the 3′ to 5′ direction.

Application

1. Alkaline elution
2. Alkaline unwinding
3. Single cell gel electrophoresis (comet assay)

Basic Information

1. Source: Recombinant protein from microorganisms
2. Molecular weight: 32.5Kd (SDS-PAGE)
3. Purity: > 90% (determined by SDS-PAGE)
4. Buffer composition: 10mM Tris-HCl, 250mM NaCl, 1mM DTT, 0.1mM EDTA, 0.15% Triton® X-100, 200µg/mL BSA, 50% Glycerol, pH 7.4 @ 25°C.
5. Enzyme activity: 10U/μL.

Unit Definition

One unit (U) is defined as the amount of enzyme required to cleave 1pmol of 34bp double-stranded oligonucleotide containing one AP site* within 1 hour at 37°C in a 10μL reaction system.

Method for AP site generation: Treat 10pmol of 34bp double-stranded oligonucleotide containing one uracil base with 1U of uracil-DNA glycosylase (UDG) at 37°C for 2 min.

Quality Control

1. Exonuclease Residue Assay: Incubate 20U of this product with 0.6μg of λ-HindIII at 74°C for 1 hour; no alteration is observed in the DNA electrophoresis band profile.
2. Endonuclease Residue Assay: Incubate 10 U of this product with 1μg of λDNA in a 20μL reaction system at 37°C for 4 hours; no alteration is observed in the DNA electrophoresis band profile.
3. RNase Residue Assay: Incubate 20U of this product with 1μg of total RNA from HeLa cells at 37°C for 30 minutes; no alteration is observed in the RNA electrophoresis band profile.
4. E. coli DNA Residue Assay: Amplify the E. coli 16S rDNA gene using ddH₂O as the template in a 50μL system. After 30 cycles, perform 1% agarose gel electrophoresis followed by staining; no amplification band is detected.

Note

This product is for research use only. It is strictly prohibited for use in human experiments.