This experiment was used to detect sequence-specific DNA-binding proteins in crude extracts.
Operation method
DNA binding protein assay
Principle
This analytical method is a simple, rapid and extremely sensitive method for the detection of sequence-specific DNA-binding proteins in crude extracts. During electrophoresis, proteins specifically bound to end-labeled DNA fragments block the migration of the fragments, thus producing clear electrophoretic bands corresponding to protein-DNA complexes.
Materials and Instruments
Plasmid DNA Move 1. Digest 10 μg of plasmid man with one or more restriction endonucleases in a volume of 100 μl to produce a DNA fragment of 25-100 bp containing the binding site with at least one end 5' protruding. Caveat 1. The length of the target DNA should be less than 300 bp to facilitate electrophoretic separation of unbound probes and protein-DNA complexes. Double-stranded synthetic oligonucleotides and restriction enzyme fragments can be used as probes in gel migration experiments. If the target protein has been identified, short oligonucleotide fragments (approximately 25 bp) should be used so that the binding site can be distinguished from that of other factors. Long restriction enzyme fragments can be used to localize protein binding sites within the putative promoter/enhancer region. The specific region of protein binding can then be analyzed at the DNA sequence level by DNaseI blotting. 2. Binding proteins for gel migration experiments can be derived from purified or partially purified proteins, or from crude nuclear and cytoplasmic extracts. Isotope-labeled DNA or RNA must also be prepared. generally, DNA nucleotide probes are end-labeled with g-32P and T4 polynucleotide kinase, and isotope-labeled RNA is synthesized in vitro using phage RNA polymerase and isotope-labeled nucleotides. The components required for the binding reaction are a salt-containing solution (magnesium chloride, sodium chloride, or potassium chloride), a buffer system (Tris-HCl or HEPES), a reducing agent (DTT), glycerol, nonspecific competing DNA (poly(dI:dC)(dI:dC)), and possibly a nonionic decontaminant. After binding proteins and isotope-labeled probes, the complexes are separated by electrophoresis on nondenaturing polypropylene gels, which are subsequently dried and radiographically autoradiographed, or analyzed by PhosphorImage.3 The gel migration assays are based on the theoretical theory of the migration of DNA from a polypropylene gel. 3. Gel migration experiments are theoretically simple and fast, but for successful gel migration experiments, a number of parameters need to be optimized, which are mainly influenced by the origin of the binding protein and the characteristics of the probe binding site. The following factors need to be optimized: preparation of the extraction solution (nuclease and phosphatase contamination will degrade the probes), concentration of binding proteins, concentration of probes, concentration of non-specific probes, formulation and pH of the buffer, characteristics of the polypropylene gel electrophoresis and electrophoretic conditions, holding times and temperatures, carrier proteins, and presence or absence of cofactors (e.g., metal ions such as zinc, or cadmium, or hormones). In summary, the total volume of the reaction should be minimal (20uL). For more product details, please visit Aladdin Scientific website.
dNTP DNA polymerase Klenow enzyme TE TAE ethidium bromide TEMED ammonium persulfate BSA
Incubators Centrifuge tubes
2. Add 100 μCi [ α-32P ] dNTP, 4 μl 5 mmol/l mixture of the three NTPs, and 2.5 U Klenow enzyme. Incubate for 20 min at room temperature.
3. Add 4 μl of dNTP solution containing 5 mmol/l of the same dNTP as labeled and incubate for 5 min at room temperature.
4. Precipitate DNA and dissolve in TE.5. Add 10× spiking buffer and electrophoresis with a 2% mini agar gel (containing ethidium bromide 5 μg/ml) in TAE or TBE buffer.6. Observe the electrophoretic bands under a long-wave transmitted UV lamp. Cut a parallel slit downstream of the band and insert a piece of DEAE membrane (wetted in gel buffer first). Squeeze the membrane tightly into the gel and continue electrophoresis until the swim moves to the membrane.
7. Wash the membrane in electrophoresis buffer and blot dry on filter paper. Place the membrane into the bottom of a round-bottomed tube with a 1.5 ml screw cap containing 400 μl of DEAE eluent, and water bath at 68°C for 30 min.
8. Transfer the eluate into a 1.5 ml centrifuge tube and microcentrifuge at 4°C for 15 min. Transfer the supernatant into a clean tube, leaving 10 μl at the bottom of the original tube.9. Add 4 μl of 1 mol/l MgCl2 to the supernatant. precipitate the DNA with 1 ml of 100% ethanol and resuspend in 100 μl of TE buffer.10. 1 μl was taken at cpm/μl min and the DNA concentration was estimated by quantification using the ethidium bromide spotting method.11. Install 16 cm long electrophoretic glass plates and 1.5 mm thick spacers. Add 100 μl of 30% ammonium persulfate and 34 μl of TDMED to 40 ml of Low Ionic Strength Gel Mix, pour between the glass plates, and insert a comb with teeth ≥ 7 mm wide. The gel was allowed to polymerize for 20 min.
12. Remove the comb and bottom edge spacer. Place the gel into an electrophoresis tank containing low ionic strength electrophoresis buffer, and circulate the electrophoresis buffer with a double-ended pump at 5~30 ml/min. pre-electrophoresis for more than 90 min at 100 V (~22 mA).
13. In a microcentrifuge tube, add a final volume of 15 μl of the following: DNA probe at 10,000 c/min, BSA at a final concentration of 300 μg/ml, and approximately 15 μg of protein from the crude protein extract in buffer. Mix well and place in a 30°C water bath for 15 min.14. Add a small amount of 10× spiking buffer to 1 spiking well and allow the dye to run into the gel. Add each binding reaction solution to the other wells and electrophoresis at ~30~35mA until bromophenol blue (equivalent to ~70 bp of band) reaches the bottom of the gel.15. Remove the gel electrophoresis setup, pry off the electrophoresis glass plate, lay the glass plate with the gel adhered to it flat on the lab bench, and place three Whatman 3MM filter papers on top of the gel. Remove the filter paper along with the gel.
16. Wrap the gel in plastic film for vacuum drying. The gel film was radiographically autoradiographed overnight without the use of a sensitizing screen or radiographically autoradiographed with a mantel screen for 2-3 h. The gel was then dried under vacuum.