Single-Stranded DNA Binding Protein (SSB) preferentially binds single-stranded DNA, forming a tetramer of four identical 18.9 kDa subunits which protects 8-16 nucleotides, while not binding well to double-stranded DNA. In nature, SSB participates in DNA replication, recombination, and repair functions. In vitro, SSB has been found to stimulate certain DNA polymerase-mediated reactions by relaxing DNA secondary structure and enhancing enzyme processivity.
Source of Protein
A recombinant E. coli strain carrying the E.coli SSB gene.
50 mM Tris-HCl
200 mM NaCl
1.0 mM DTT
0.1 mM EDTA
pH 7.5 @ 25°C
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 27,880 and molecular weight of 18,975 Daltons.
SDS-Page (Physical Purity Assessment)
2.0 µL of concentrated 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 99% 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 µµL 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 µg 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.
DNA Binding Assay
Single-stranded DNA binding ability was confirmed in a PCR inhibition assay by adding decreasing amounts of E.coli SSB to a series of PCR reactions containing target DNA, 200 µM dNTPs, 1X PCR buffer and Taq DNA Polymerase. Reactions were incubated in a thermal cycler and subjected to 25 PCR cycles. Samples were resolved using agarose gel electrophoresis and amount of SSB required to block 100% accumulation of PCR product was recorded. Acceptance criteria for assay: 0.70 µg E.coli SSB is required to inhibit PCR amplification of 5 ng target DNA following 25 cycles of PCR.
|E. coli Single-Stranded DNA Binding Protein|
|Unit Size||1.5 mg|
|SDS||Available on request|
|Storage Temperature||-25 to -15°C|
|SS Exonuclease||25 µg <1.0% release|
|DS Exonuclease||25 µg <1.0% release|
|DS Endonuclease||25 µg = No conversion|
|DNA Binding Assay||0.7 µg inhibits PCR|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
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.
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