What is the best or common approach by which we can detect co-factors (activators and repressors) of a nuclear receptor (e.g. androgen receptor) in the nucleus of a cancer cell? Any help would appreciate.
If these nuclear factor are DNA binding factors, then you can characterise these into activating targets and repressing targets, These binding consenescence of targets can be pull down (may be by biotin-streptavidin magnetic pull down) along with its bound factor. Further novel targets can be identified using mass-spectrometry.
Co-factors can bind directly to AR or could bind to the DNA sequences surrounding the AR binding sites. Either way, identifying such factors is not an easy task. For AR interacting proteins, purification of AR and its associated proteins can be done by biochemical means, flag epitope tags and flag peptide elutions are a popular means. The eluted AR and associated proteins are then characterized by mass spec. For a specific AR target sequence, you could try a pull down using the DNA sequence and nuclear lysates from AR expressing cells. The trouble with DNA pull downs is removing all of the non-specific DNA binding proteins from the lysates. In any case, you need to start with a lot of material and reduce the background, an enrichment of 1000-10,000 fold is usually required in order to get enough material for MS. Talk to a real biochemist for advice.
Hi,
Please check this article.
Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene.
Ou XM, Lemonde S, Jafar-Nejad H, Bown CD, Goto A, Rogaeva A, Albert PR.
J Neurosci. 2003 Aug 13;23(19):7415-25.
Good Luck
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