DIG Gel Shift Kit, 2nd generation

Description

• General description: DIG Gel Shift Kit, 2nd generation contains reagents for nonradioactive 3′-end labeling of oligonucleotides, to be used in a "gel mobility shift" assay. It also contains offers, electrophoresis reagents and an enzyme-labeled antibody to facilitate the detection of DNA-protein complexes.DIG Gel Shift Kit employs recombinant terminal transferase and digoxigenin (DIG)-11- dideoxyuridine triphosphate (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.
• Application: DIG Gel Shift Kit, 2nd generation has been used for nonradioactive detection of sequence-specific DNA binding proteins. The DIG Gel Shift Kit is used to prepare nonradioactive, 3′-end labeled oligonucleotide probes to detect DNA-protein complexes in a "gel mobility shift assay".[1][2]
• 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).
• Packaging: 1 kit containing 16 components. 1 kit for up to 20 Oligonucleotide 3-end labeling reactions with DIG-11-ddUTP, 200 binding reactions, chemiluminescent detection reaction for up to 20 blots, DNA binding protein and oligonucleotide for up to 20 control reactions
• Physical form: Chlorophenol red-β-D-galactopyranoside, monosodium salt
• Preparation Note: 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.SampleAmount: 3.85pmol or 100ngType: 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.Time requiredOligonucleotide annealing and labeling: 10minutesFormation of oligonucleotide-protein complexes: 25minutesElectrophoresis: 1 hour to overnight, depending on electrophoresis systemBlotting: 1 - 2 hoursImmunological detection: 2hoursExposure to X-ray film or imager: 15 - 40minutes
• Other Notes: For life science research only. Not for use in diagnostic procedures.