T4 Gene 32 Protein

• Stablize single-stranded DNA regions
• Increases processivity of some DNA polymerases

T4 Gene 32 Protein is a single-stranded DNA binding protein that is required for T4 DNA replication, recombination and repair. The protein enhances the performance of several DNA synthesis-related activities, including DNA sequencing of regions that are rich in secondary-structure and PCR amplification. T4 Gene 32 also greatly stimulates the rate of synthesis of T4 DNA Polymerase on primed-single-stranded substrates (5–10 fold increase in synthesis rate).

This enzme is supplied in 20 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, 0.5 mM DTT and 50% glycerol; pH 8.0 at 25°C.

Ask about glycerol free options

SDS is available upon request.

Storage temperature: –25°C to –15°C

Molecular weight: 33,506 Daltons

Source of recombinant enzyme protein
The protein is produced by a strain of E. coli that overexpresses the gene 32 protein from bacteriophage T4.

References

1. Chase, J.W., et al. (1986) Ann. Rev. Biochem. 55:103.
2. Sinha, N.K., et al. (1971) J. Mol. Biol. 62:267.
3. Krisch, H.M., et al. (1974) J. Mol. Biol. 88:89.
4. Gold, L., et al. (1976) J. Biol. Chem. 251:7251.
5. Delius, H., et al. (1972) J. Mol. Biol. 67:341.
6. Brack, C., et al. (1975) J. Mol. Biol. 96:6932.
7. Huberman, J. A., et al.(1971) J. Mol. Biol. 62:39.
8. Baugh, L.R., et al. (2001) Nucleic Acids Res. 29 (5), E29.

Quality control analysis

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

The ability of Gene 32 Protein to bind single-stranded DNA was measured using a gel-shift assay with a single-stranded, fluorescently labeled oligonucleotide. Serial dilutions of the enzyme were made in 1X T4 GP32 reaction buffer (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate and 1mM DTT; pH 7.9) and added to 10 µl reactions containing a 5ʹ-FAM labeled oligonucleotide substrate and 1X T4 GP32 Reaction Buffer. Reactions were incubated 20 minutes at 37°C, placed on ice, and run on a 15% TBE urea gel. DNA binding ability is observed as a band shift in the apparent molecular weight of the oligonucleotide on the TBE-urea gel.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Sequencing
• PCR amplification