Talk:Knight:Protein DNA binding/Option 1

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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. . pmid:8303274. PubMed HubMed [Rebar-Science-1994]

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

  1. Greisman HA and Pabo CO. . pmid:9005850. PubMed HubMed [Greisman-Science-1997]

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

  1. Pomerantz JL, Wolfe SA, and Pabo CO. . pmid:9467467. PubMed HubMed [Pomerantz-Biochem-1998]

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

  1. Wolfe SA, Ramm EI, and Pabo CO. . pmid:10903945. PubMed HubMed [Wolfe-Structure-2000]
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