T4 Gene 32 Protein is a single-stranded DNA binding protein which is required for T4 DNA replication, recombination and repair. The protein has exhibited an ability to enhance the performance of several DNA synthesis-related activities, including DNA sequencing in secondary-structure rich regions 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).
Source of Protein
Purified from a strain of E. coli that overexpresses the gene 32 protein from bacteriophage T4.
20 mM Tris-HCl
100 mM NaCl
1.0 mM EDTA
0.5 mM DTT
pH 8.0 @ 25°C
|Storage Temperature||-25 to -15°C|
|SS Exonuclease||100 µg <1.0% released|
|DS Exonuclease||100 µg <1.0% released|
|DS Endonuclease||100 µg = No conversion|
|SS DNA Binding Activity||Functional|
|E.coli DNA Contamination||25 µg <10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
QUALITY CONTROL ANALYSIS
Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 38,690 and molecular weight of 33,506 Daltons.
SDS-Page (Physical Purity Assessment) and Specifications
2.0 µL of enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 95% purity of the concentrated sample.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µµg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate 5 µL samples of enzyme solution were 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 acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies.
DNA Binding Assay
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(50mM Potassium Acetate, 20mM Tris Acetate, 10mM Magnesium Acetate, 1mM 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 20 minutes at 37°C, plunged on ice, and run out 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.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.