Talk:Knight:Protein DNA binding/Option 1

Rebar and Pabo

 * 2.5 or 25 pM radioactive DNA fragment
 * peptide from twofold dilution series
 * 14.7&mu;g/mL poly (dI-dC)-poly (dI-dC) from Pharmacia

in degassed gel shift buffer:
 * 50mM NaCl
 * 5mM MgCl2
 * 10&mu;M 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. 150&mu;M or 300&mu;M binding site.

Protein storage
Stored at -80&deg;C
 * 2.75 mM ZnSO4
 * 50mM bis-tris propane (pH 6.8)

Reference

 * 1) Rebar-Science-1994 pmid=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
 * 20&mu;M ZnSO4
 * 100&mu;g/mL acetylated BSA
 * 5% (v/v) glycerol
 * 0.1% (w/v) NP-40
 * 2 or 4 pM of the labeled site

Incubated 1hr.

Reference

 * 1) Greisman-Science-1997 pmid=9005850

Pomerantz et al.
Total volume 10&mu;L
 * 20mM Hepes (pH 7.9)
 * 60mM KCl
 * 0.75mM dithiothreitol
 * 4% Ficoll-400
 * 300&mu;g/mL BSA

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

Protein storage

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

Reference

 * 1) Pomerantz-Biochem-1998 pmid=9467467

Wolfe et al.
Same as Greisman and Pabo except
 * Use 0.5X TBE for EMSA assays.
 * 2&mu;g/mL Ac-BSA

Protein storage
Lyophilized, resuspended in water, aliquoted and stored at -80&deg;C

Reference

 * 1) Wolfe-Structure-2000 pmid=10903945