Ultraviolet cross-linking experiments of proteins and nucleic acids
Ultraviolet cross-linking experiments of proteins and nucleic acids
Irradiation of protein-nucleic acid complexes with UV light induces covalent bond formation where nucleic acid nucleoproteins are in close contact, thus selectively labeling DNA-binding proteins that bind specifically to DNA recognition sites. Because of the transfer of the marker from nucleic acid to protein, the molecular mass of DNA-binding proteins in crude mixtures can be determined quickly and reliably. Source: Compact Molecular Biology Laboratory Guide, Fifth Edition
Operation method
UV cross-linking of probes substituted with bromodeoxyuridine (BrdU)
Principle
DNA containing thymidine halides such as bromodeoxyuridine (BrdU) is more sensitive to UV-induced cross-linking than non-substituted DNA. UV crosslinking efficiencies in this protocol are 0.1% to 10%, and it is rare to have more than 1 crosslinking event in a single complex.
Materials and Instruments
M13 single-stranded vector containing the binding site under study Move 1) 10 μL of single-stranded M13 vector containing the target sequence with a high-affinity protein binding site was mixed with equimolar 17 bp M13 universal primer. The final volume was adjusted to 100 μL with 1× restriction endonuclease buffer (50 mmol/L NaCl). heated at 90°C for 5 min and cooled overnight at room temperature. 2) Add the following reactants to the hybridization mixture: 50 μL [ α32P ] dCTP (3000 Ci/mmol) 3.5 μL 50× dNTP/BrdU solution 1.75 μL 0.1 mol/L dithiothreitol (DTT) 7.5 μL 10× restriction endonuclease buffer (NaCl final concentration 50 mmol/L) 7 μL H2O 5 μL Klenow enzyme (25 U) incubate at 16°C for 90 min. 3) Heat at 68°C for 10 min to inactivate Klenow enzyme. 4) Digest with 40 U restriction endonuclease under appropriate conditions to produce a DNA fragment of 20 ~ 600 bp. 5) Add ammonium acetate to 0.3 mol/L, precipitate DNA with 2x volume of 100% ethanol and resuspend in TE buffer. 6) Electrophoresis on an agarose gel containing 0.5 μg/mL ethidium bromide. Separate the desired DNA fragments with a DEAE membrane. 7) Detect the specific activity of BrdU-substituted DNA fragments with a scintillation counter and estimate the concentration of DNA by quantification with ethidium bromide dot blotting. Probe integrity and function can be assayed by mobility shift DNA binding assay. 8) The following binding reactions were established in 1.5 mL round bottom vials: 105 cpm equilibrium-labeled probe, buffered extract containing binding protein, and 10-20 μg of DNA carrier such as poly (dl-dC) - poly (dl-dC). The total volume was adjusted to 50 μL. The vials were sealed with plastic film and fixed with Parafilm. 9) Place the vials on a test tube rack and irradiate them with an inverted UV transilluminator (305 nm, 7000 μW/cm2 ) 5 cm directly above the vials for 60 min. 10) Add the following reactants to each binding reaction tube: 1 μL 0.5 mol/L CaCl2 1 μL of 0.5 mol/L CaCl2 4 μg of DNAase I 1U micrococcal nuclease Digestion at 37°C for 30 min. 11) Add an equal volume of 2 x SDS sample buffer to the reaction mixture and boil at 100°C for 5 min. 12) Electrophoresis of the samples, including 14C-labeled protein molecular mass markers, was performed on discontinuous SDS-polyacrylamide gels at appropriate concentrations. 13) At the end of electrophoresis, cut away the dye from the gel front. 14) The gel is dried and autoradiographed with a sensitized screen for 1 to 3 days to observe the cross-linked proteins. Common Problems Other reagents required: 25 U Escherichia coli DNA polymerase I of Klenow fragment, ammonium acetate. 100% ethanol. TE buffer, buffered with DNA Binding Protein Extract, Macromolecular Carrier DNA such as poly (dl-dC) poly (dl-dC) poly (dl-dC) poly (dl-dC). 0.5 mol/L CaCl2 poly (dl-dC) - poly (dl-dC) DNA enzyme I (Worthington) (Worthington) 1 U Micrococcus ribozyme ( Worthington) Worthington 2 × SDS sample buffer. Fluor (Du Pont NEN Enhance or equivalent). 14 C Labeled protein molecular mass marker For more product details, please visit Aladdin Scientific website.
17 bp M13 universal primer 1× and 10× restriction endonuclease buffer (containing 500 μmol L and 500 mmol L NaCl, respectively) 3000 Ci mmol [α32P] dCTP 50× dNTP BrdU solution 0.lmol L dithiothreitol (DTT)
DEAE membrane (Schleicher &- Schuell NA45) 0.5 mL round-bottomed vials with screw caps (Nunc or equivalent) Water bath UV transilluminator