T4 Gene 32 Protein

• Stabilizes single-stranded regions of DNA
• Enhances DNA sequencing procedures in secondary structure rich regions
• Increases efficiency of PCR amplification
• Stimulates synthesis rate of T4 DNA Polymerase on primed-single-stranded substrates

The native Gene 32 Protein from bacteriophage T4 (T4gp32) is a single-stranded DNA binding protein that is required for T4 DNA replication, recombination and repair.

The T4 Gene 32 Protein has exhibited an ability to enhance the performance of several DNA synthesis-related activities in secondary-structure rich regions, including PCR amplification and DNA sequencing. Improved yields and quality of templates may be achieved with the use of DNA-binding proteins in amplification and sequencing reactions. The T4 Gene 32 Protein also stimulates the rate of synthesis of T4 DNA Polymerase on primed-single-stranded substrates showing a 5–10-fold increase in synthesis rate.

The product is supplied in 20 mM Tris-HCl, 100 mM NaCl, 0.5 mM DTT, 1 mM EDTA, 50% glycerol; pH 8.0 at 25°C.

The T4 Gene 32 Protein is a single-stranded nucleic acid binding protein that has the function of stabilizing single-stranded regions of DNA. The ability of T4 Gene 32 Protein to enhance the performance of several DNA synthesis-related activities is based on its essential function in the replication of bacteriophage T4.

Gp32 binds transiently and cooperatively to ssDNA template sequences as these entities are exposed by the processive helicase operating within the replication complex. This binding puts these ssDNA sequences into optimal conformations for interacting with DNA polymerases and other replication proteins. By coating these transiently exposed ssDNA sequences, gp32 also protects them from degradation by nucleases while they discharge their templating (and other) functions in association with the leading- and lagging-strand DNA polymerases.

Instructions for using T4 Gene 32 Protein are provided in the corresponding kit protocol in the resources below.

Quality Control

DNA binding of single stranded DNA by T4 Gene 32 Protein 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, 1 mM DTT; pH 7.9) and added to 10 μL reactions containing a 5’-FAM–labeled oligonucleotide substrate, and 1X T4 GP32 Reaction Buffer. Reactions were incubated for 20 minutes at 37°C, plunged on ice, and run out on a 15% TBE-Urea gel. DNA binding ability was observed as a band shift in the apparent molecular weight of the oligonucleotide on the TBE-Urea gel.

Protein concentration (OD₂₈₀) of T4 Gene 32 Protein was determined by OD₂₈₀ absorbance.

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

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

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

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

E. coli contamination was evaluated using 5 μL replicate samples of protein solution 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.

This product is available for molecular biology applications such as:

• Increasing the efficiency of enzymes, such as Taq DNA polymerase, reverse transcriptase and telomerase
• Promoting DNA synthesis by DNA polymerase
• Reducing the effect of inhibitory substances in PCR
• Enhancing site-specific mutagenesis
• Eliminating extra bands during sequencing
• Marking single stranded DNA for electron microscopy