Talk:Knight:Protein DNA binding/Option 1

DNA binding reaction conditions from the literature

Rebar and Pabo

• 2.5 or 25 pM radioactive DNA fragment
• peptide from twofold dilution series
• 14.7μg/mL poly (dI-dC)-poly (dI-dC) from Pharmacia

in degassed gel shift buffer:

• 50mM NaCl
• 5mM MgCl₂
• 10μM ZnSO₄
• 5% glycerol
• 0.1mg/mL BSA
• 0.1% NP-40
• 15mM HEPES (pH 7.8)

Equilibrated at room temperature for 30 mins or 4 hours.
Electrophoresed on 10% polyacrylamide gels in 0.03 M tris-Hepes (pH 7.8).
Used freshly thawed peptide.
150μM or 300μM binding site.

Protein storage

• 2.75 mM ZnSO₄
• 50mM bis-tris propane (pH 6.8)

Stored at -80°C

Reference

1. Rebar EJ and Pabo CO. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science. 1994 Feb 4;263(5147):671-3. DOI:10.1126/science.8303274 |

Greisman and Pabo

Similar to Rebar and Pabo

• 15mM Hepes-NaOH (pH 7.9)
• 50mM KCl
• 50mM potassium glutamate
• 50mM potassium acetate
• 5mM MgCl₂
• 20μM ZnSO₄
• 100μg/mL acetylated BSA
• 5% (v/v) glycerol
• 0.1% (w/v) NP-40
• 2 or 4 pM of the labeled site

Incubated 1hr.

Reference

2. Greisman HA and Pabo CO. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science. 1997 Jan 31;275(5300):657-61. DOI:10.1126/science.275.5300.657 |

Pomerantz et al.

• 20mM Hepes (pH 7.9)
• 60mM KCl
• 0.75mM dithiothreitol
• 4% Ficoll-400
• 300μg/mL BSA

Total volume 10μL

Incubated at 30°C for 40 mins.
Resolved on 4% nondenaturing polyacrylamide gels using Tris-glycine electrophoresis buffer.
Titrated DNA-binding sites in the 1-50μM concentration range.
Protein concentration exceeded DNA concentration always by >=10X.

Protein storage

• 50mM Tris (pH8.0)
• 100mM KCl
• 10% glycerol

Reference

3. Pomerantz JL, Wolfe SA, and Pabo CO. Structure-based design of a dimeric zinc finger protein. Biochemistry. 1998 Jan 27;37(4):965-70. DOI:10.1021/bi972464o |

Wolfe et al.

Same as Greisman and Pabo except

• Use 0.5X TBE for EMSA assays.
• 2μg/mL Ac-BSA

Protein storage

Lyophilized, resuspended in water, aliquoted and stored at -80°C

Reference

4. Wolfe SA, Ramm EI, and Pabo CO. Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers. Structure. 2000 Jul 15;8(7):739-50. DOI:10.1016/s0969-2126(00)00161-1 |