The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein-DNA complexes.
Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone-DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.
Adkins
Nicholas L
NL
Marshall University, Department of Biological Sciences Byrd Biotechnzology Science Center, 1 John Marshall Drive, Huntington, WV 25755, United States.
Hall
J Adam
JA
Georgel
Philippe T
PT
D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000737:chemistry; D004275:DNA, Ribosomal / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D066298:In Vitro Techniques; D047331:Lytechinus / Q000737:chemistry / Q000235:genetics; D046911:Macromolecular Substances / Q000032:analysis / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*
D002843:Chromatin; D004275:DNA, Ribosomal; D046911:Macromolecular Substances; D009707:Nucleosomes; D011506:Proteins