顺式调控元件的寻找20121116.pptVIP

分子生物学工作基础 基因的表达调控刘喆zheliu@tmu.edu.cn Application: EMSA: Principle Preparation of Nuclear and Cytosolic Extracts The Probe Labeling the Probe Removal of free radioactive material Reagents Analysis by non-DenaturingPolyacrylamide Gel Electrophoresis 准备非变性PAGE凝胶 Proof of Specificity R. Voll 09/01 上样 电泳 干胶 放射自显影 R. Voll 09/01 Supershift using antibodies against the DNA-binding protein Competition for binding to the radiolabeled probe using unlabeled wildtype and mutated oligos R. Voll 09/01 Supershift A double-stranded oligonucleotide containig a NF-?B- binding site is labelled radioactive and incubated with a nuclear extract. During gel-electrophoresis, NF-?B bound to the oligonucleotide causes a shift compared to the free probe. p50/p65 Free probe Radioactive labelled oligonucleotide with NF-?B - binding site (probe) and bound NF-?B Radioactiv labelled oligonucleotide with NF-?B - binding site (probe) Nuclear extract of activated cells Nuclear extract of activated cells with anti-p50 antibody p50/p65 + anti-p50 R. Voll 09/01 Competition with Unlabeled Oligos Increasing amounts of unlabeled oligos containing the NF-kB binding site or unlabeled oligos with a mutated binding site were added to the reaction mix prior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo. p50/p50 Free probe p50/p65 Unspecific Wild type oligo Mutated oligo GGG GAC TTT CCC GGA GAC TTT CCC * Chromatin packing occurs on several levels. This schematic drawing shows some of the orders of chromatin packing thought to give rise to the highly condensed mitotic chromosome * * Chromatin packing occurs on several levels. This schematic drawing shows some of the orders of chromatin packing thought to give rise to the highly condensed mitotic chromosome * * Chromatin packing occurs on several levels. This schematic drawing shows some of the orders of chromatin packing thought to give rise to the highly condensed mitotic chromosom