UNSPSC Code: 12352200
Components: Labeling Buffer 5x concentrated; CoCl<SUB>2</SUB> Solution 25 mM; DIG-ddUTP Solution, 1 mM Digoxigenin-11-ddUTP; Recombinant Terminal Transferase 400 U/μl; Binding Buffer 5x concentrated; Control Oligonucletide ds 39mer, unlabeled 0.1 μg/μl, 3.85 pmol/μl; DIG-labeled Control Oligonucleotide ds 39mer 0.4 ng/μl, 15.54 fmol/μl; Control Factor Oct2A 25-75 ng/μl; Poly [d(I-C)] 0.1 μg/μl; Poly [d(A-T)] 0.1 μg/μl; Poly L-lysine 0.1 μg/μl; Loading Buffer without bromophenol blue; Loading Buffer with bromophenol blue; Anti-Digoxigenin-AP 750 U/ml; CSPD 10 mg/ml; Blocking Reagent
RIDADR: UN 3316 9
Features and Benefits:
The electrophoresis assay works best with shorter (30 to 100bp) DNA fragments, since these are less likely to have sequences that are outside the specific binding site, but can interact nonspecifically with target proteins.
• Convenient, since the technique does not require special equipment or technology.
• Reproducible, since DIG-labeled probes are stable indefinitely.
• Safe, because the assay is nonradioactive.
• Reliable, because the kit provides DIG-labeled control oligonucleotides to establish that the assay is working.
• Function-tested with the controls provided in the kit (See "Quality").
• Sensitive, since the kit can detect as little as 20fmol of the control oligonucleotide (after it is labeled according to the kit protocol).
DIG Gel Shift Kit, 2nd generation is used for nonradioactive detection of sequence-specific DNA binding proteins. The DIG Gel Shift Kit is used to prepare nonradioactive, 3â€²-end labeled oligonucleotide probes to detect DNA-protein complexes in a "gel mobility shift assay".
Sigma Life Science is committed to bringing you Greener Alternative Products, which adhere to one or more of The 12 Principles of Greener Chemistry. This product is designed as a safer chemical. The DIG (digoxigenin) System was established as a sensitive and cost-effective alternative to using radioactivity for the labeling and detection of nucleic acids. There are many available publications that prove the versatility of the DIG System, so use of radio-labeling is no longer the only option for labeling of DNA for hybridization.
The kit contains reagents for nonradioactive 3′-end labeling of oligonucleotides, to be used in a "gel mobility shift" assay. It also contains controls, electrophoresis reagents, and an enzyme-labeled antibody to facilitate the detection of DNA-protein complexes.
Note: The combination of recombinant Terminal Transferase and DIG-11-ddUTP makes the labeling reaction very flexible. It can be used to label the 3′ ends of any oligonucleotide (whether it has a 5′- overhanging end, a 3′-overhanging end, or blunt ends). Both single- and double-stranded DNA can be labeled.
For life science research only. Not for use in diagnostic procedures.
Chlorophenol red-β-D-galactopyranoside, monosodium salt
The study of DNA-protein interactions has been greatly facilitated in recent years by the rapid and simple "gel retardation" or "gel mobility shift" assay. Because free DNA and DNA-protein complexes migrate differently during gel electrophoresis, they can be separated and detected on native (nondenaturing) polyacrylamide or agarose gels.
Amount: 3.85pmol or 100ng
Type: Oligonucleotides with 5′-overhanging ends, 3′-overhanging ends, or blunt ends; single- or double-stranded DNA fragments (30 - 200bp)
Note: Ideally, fragments to be labeled should be between 30 and 100bp.
Oligonucleotide annealing and labeling: 10minutes
Formation of oligonucleotide-protein complexes: 25minutes
Electrophoresis: 1 hour to overnight, depending on electrophoresis system
Blotting: 1 - 2 hours
Immunological detection: 2hours
Exposure to X-ray film or imager: 15 - 40minutes