Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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DNA Band Shift Assay |
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| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley | ||||||||||||||||||||||||||||||||||||||||||
| Overview |
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| The protocol is designed to detect protein-DNA complexes. The DNA is radiolabeled then mixed with the protein. The protein binding can be detected because the protein-DNA complexes have a slower mobility in a polyacrylamide gel than the free DNA. It has worked successfully for the KP, δ-COO, and PN88 proteins. | |||||||||||||||||||||||||||||||||||||||||||
| Procedure |
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A. Making the Band Shift Probe 1. Digest the plasmids containing regions of DNA to be used as the probes with the appropriate Restriction Enzymes (see Hint #2). 2. End label the DNA fragments with γ-[32P]-ATP (see Protocol ID# 552) (CAUTION! See Hint #1). 3. Gel-purify the labeled DNA fragment (see Protocol ID#1265). B. Binding Reaction 1. Combine the following (see Hint #3): (putative) DNA-binding Protein 1,000 to 5,000 cpm of radiolabeled DNA (from Section A) 4 μl of 5X Reaction Binding Buffer ddH2O to a final volume of 20 μl 2. Set up a series of reaction tubes with increasing amounts of protein (up to 10 pmole) to titrate the amount of protein needed to shift the free probe. 3. Incubate for 60 min at room temperature. C. Electrophoresis of Shifted DNA 1. Prepare a 4% Polyacrylamide Gel (see Protocol ID#1272): 6.7 ml of 30% Acrylamide Solution 1.25 ml of 20X TBE 500 μl of 10% Igepal CA-630 41.6 ml of ddH2O Just before pouring the gel add: 250 μl of 10% Ammonium Persulfate 60 μl of TEMED 2. Use 1.5 mm spacers between the gel plates. 3. After the gel has polymerized, allow the gel to cool at 4°C in a cold room. 4. Add 1 μl of 6X Loading Dye to 5 μl of Protein:DNA mixture. 5. Load the Protein:DNA mixture onto the gel. 6. Electrophorese the Protein:DNA mixture at 4°C and 150 V in 0.5X TBE until the Bromophenol Blue dye travels approximately one-half to three-quarters the distance to the bottom of the gel. 7. Disassemble the gel and transfer it to solid support for drying (e.g., Whatman 3MM paper). 8. Dry the gel. 9. Autoradiograph the gel to analyze shifted protein:DNA complexes. |
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| Solutions |
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| BioReagents and Chemicals |
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HEPES Restriction Enzymes EDTA Sodium Chloride Polydeoxyinosinic-Deoxycytidylic Acid TEMED Ammonium Persulfate Boric Acid Acrylamide Bis-acrylamide Sodium Hydroxide Glycerol Tris I3-[32P]-ATP Xylene Cyanol FF Bromophenol Blue PMSF IGEPAL CA-630 DTT |
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| Protocol Hints |
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. For P-element band shifts, use probes that correspond to the ends of the P-element. 3. For other proteins, you can adjust the incubation time, salt concentration, amount of competitor DNA, and different detergents. |
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| Citation and/or Web Resources |
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1. Mobility shift DNA-binding assay using gel electrophoresis. Current Protocols in Molecular Biology, pp. 12.2.1-12.2.10 |
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