凝胶阻滞,EMSA

1、YTH R. Voll 09/01A double-stranded oligonucleotide containig a NF- B- binding site is labeled with a radioactive isotope and incubated with a nuclear extract.During gel-electrophoresis, NF- B bound to the oligonucleotide causes a shift compared to the free probe.NF- BFree ProbeRadioaktively labeled

2、oligonucleotidewith NF- B - binding site (probe) and bound NF- B Radioactively labeled oligonucleotide with NF- B - binding site (probe)Nuclear extract of non-activated cellsNuclear extract ofactivated cells R. Voll 09/01SupershiftA double-stranded oligonucleotide containig a NF- B- binding site is

3、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/p65Free probeRadioaktiv labelled oligonucleotidewith NF- B - binding site (probe) and bound NF- B Radioactiv labelled oligonucleotide

4、 with NF- B - binding site (probe)Nuclear extract of activated cellsNuclear extract of activated cells with anti-p50 antibodyp50/p65 + anti-p50R. Voll 09/01Competition with Unlabeled OligosIncreasing amounts of unlabeled oligos containing the NF- B binding siteor unlabeled oligos with a mutated binding site were added to the reaction mixprior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo.p50/p50Free probep50/p