Gel blocking assay of DNA binding proteins
Gel blocking assay of DNA binding proteins
The gel-blocking assay for analyzing DNA-binding proteins was invented by Fried and Crothers (1981) and was the first to provide a dynamic method for analyzing the interaction of Escherichia coli lactose repressors with their DNA-binding sites. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Gel blocking assay of DNA binding proteins
Materials and Instruments
Nuclear extract or protein fraction as control Nuclear extract or protein fraction Move makings For more product details, please visit Aladdin Scientific website.
Ficoll 400 polyvinyl alcohol sucrose staining solution 10X Tris-aminoacetic acid or Tris-boric acid-EDTA (TBE) buffer 4%~7% neutral polyacrylamide gel poly(dI-dC) 32P-labeled control DNA 32P-labeled target DNA
Hybridization membrane
Buffers and solutions
See Appendix 1 for the composition of storage solutions, buffers and reagents.
The storage solution needs to be diluted to the appropriate concentration.
Ficoll 400 (20% m/V)
Ficoll is dissolved in sterile water, dispensed into 100ul and frozen at -20°C.
Polyvinyl Alcohol (10% m/V)
Polyvinyl Alcohol is dissolved in sterile water, dispensed in 100 ul and frozen at -20°C.
Sucrose Staining Solution
0.25% (m/V) bromophenolan
0.25%(m/V)xylene cyanide
40% (m/V) Sucrose purification (glucose)
Dissolve in water and filter through 0.22um filter; store at room temperature or 4°C.
10X Tris-aminoacetic acid or Tris-boric acid-EDTA (TBE) buffer
Gel
4%~7% neutral polyacrylamide gel (thickness ≤1.5 mm)
Nucleic acids and oligonucleotides
poly(dI-dC)(lmg/ml)
Dissolve appropriate amount of poly(dI-dC) in sterile water, dispense 100 ul per tube and freeze at -20°C. For more information on the role of poly(dI-dC) in gel block assays please see the poly(dI-dC) section of the letterboxing section.
Radioactive compounds
32P-labeled control DNA
32P-labeled target DNA >20bp [specific activity ≥2. 5x107cpm/ton (≥5000cpm/fmol)]
DNA fragment labeling can be accomplished by phosphorylation (Chapter 9, Schemes 13-16, or Chapter 10, Scheme 2); complementation of 3'-retracted ends with DNA polymerase (Chapter 9, Scheme 10, or Chapter 10, Scheme 7); or seeding of radiolabeled nucleotides into the probe by PCK (Chapter 8, Scheme 1). This last method is the fastest and provides the highest specific activity of the probe For advice on how to label DNA probes, see the section at the end of this protocol: Troubleshooting and Optimization of Gel Block Assays.
Special equipment
Hybridization membrane
Cells and Tissues
Nuclear extracts or protein fractions as controls
Nuclear extracts or protein fractions
Extracts are prepared by any of the methods described in Scheme 1. Proteins obtained by in vitro translation in rabbit reticulocyte lysates or wheat germ extracts can also be used as DNA binding proteins. In the latter case, 1 to 20 ul of protein is usually used instead of nuclear extracts. 
Methods
1. In a sterile 1.5 ml centrifuge tube add.
32P-labeled target DNA 1ng (1~10fmol)
1 mg/ml poly(dI-dC) 1ul
Nuclear extract (5~10ug) ≤10ul
or
Protein fraction ≤10ul
20% Ficoll 400 5ul
or
10% Polyvinyl Alcohol 4ul
H2O to 20ul 
2. Centrifuge the reaction tube for a few seconds to allow the reaction mixture to settle to the bottom of the tube. Ice bath for 10-30 min.
3. Add 3ul of sucrose stain to each tube. The sample is added to a 4% to 7% neutral polypropylene amide gel.
4. Run the gel in 0.5XTris-Glycine buffer or 0.5XTBE buffer, 200~250V, 20mA, ≥ 2 h.
Depending on the instability of the binding protein and the affinity of the binding reaction, it may sometimes be necessary to run the gel at 4°C.
5. After electrophoresis is complete, pry off the gel plate, transfer the gel to a piece of sturdy hybridization membrane, and dry the gel in a gel dryer for about 1 h.
6. Expose the X-ray film at -20°C for ≥1 h with the dry gel to visualize the radiolabeled DNA fragments. Lower abundance DNA-protein complexes can be detected by phosphorimage analysis in about 1-3 hours. 
