ChromoBlog

Catch them if you can: Novel Nano-Traps for „old“ molecules

Posted by Christoph Eckert on Aug 4, 2014 3:01:39 PM

It was discovered 35 years ago, was awarded the title „molecule of the year“ in 1993 by the journal Science, and has amassed a whopping 72,000 research papers to date. Hardly any other protein has stirred up so much scientific interest as the transcription factor with the rather unspectacular name p53, which became famous as a tumor suppressor (1). It patrols the nucleus for damaged DNA and prevents cells from dividing or, if the damage is too large, induces apoptosis. Thus, p53 can be regarded as a kind of emergency brake that is pulled when something goes utterly wrong. Somewhat more poetically, Sir David P. Lane, probably the best known researcher in this field and the second most cited UK-based scientist in the 1990’s, called it the „guardian of the genome“ (2).

wordle13-3

This guardian molecule is, in turn, controlled by other guardians, most notably Mdm2 and Mdm4 (the latter also known as MdmX or HdmX), which affect both the transcriptional activity and the protein level of p53. Together, these proteins form part of a „circuit board“ that controls the cell cycle and apoptosis (3). Originally identified as a p53 inhibitor, Mdm4 has recently been demonstrated to exert more complex effects on p53 depending on the specific growth conditions (4). As it has been shown that Mdm4 amplification and/or over-expression occurs in several diverse tumors, it has become a highly interesting drug target of its own.

Mdm4_WesternTo better understand the intricate network of p53 regulation, first and foremost during tumor development, the focus of current research is shifting more and more from genetic to biochemical analyses (5). In order to facilitate this work, ChromoTek has recently launched two novel Nano-Traps. These two binding proteins specifically recognize and bind to p53 or Mdm4, respectively, allowing fast (<30 min) and clean one-step pull-downs of these molecules from cell or nuclear extracts and subsequent characterization of interacting proteins that are bound to them. The eluates can then be further analyzed by a variety of different techniques such as Western, ELISA, mass spectrometry etc. 

p53_WesternBoth the p53-Trap and the Mdm4-Trap are derived from the variable part of a heavy chain antibody from the variable part of a heavy chain antibody from Alpaca, a so-called VHH.With a molecular weight of only about 15 kD, VHHs are among the smallest intact fully functional antibodies. They are extremely resistant to heat, salt and high/low pH and can reach subnanomolar affinities. Unlike conventional antibodies, they don’t possess light or heavy chains that can dissociate and lead to contaminants that can give a high background in IP experiments. As recombinant proteins they can be produced in constant quality with virtually no batch-to-batch variation. Like most of our Nano-Traps, the p53-Trap and the Mdm4-Trap have been optimized by genetic engineering to achieve both high specificity and high expression. For direct use in IP experiments, they are coupled to a monovalent matrix (agarose particles).

The p53-Trap binds the N-terminal epitope from aa 1 to 81 of p53 and has been shown to work with the human protein. The Mdm4-Trap recognizes the epitope from aa 1 to 129 of Mdm4 and has been tested with human, mouse and hamster homologs.

Free test sample for p53 or Mdm4

Selected literature

  1. DeLeo AB, Jay G, Appella E, Dubois GC, Law LW, Old LJ.
    Proc Natl Acad Sci USA 1979 76(5): 2420-4.
    Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse.
  1. Lane DP, Nature 1992 Jul 2;358(6381):15-6.  
    p53, guardian of the genome.
    Comment in Checkpoint policing by p53.[Nature. 1992]
  1. Sullivan KD1, Gallant-Behm CL, Henry RE, Fraikin JL, Espinosa JM. Biochim Biophys Acta. 2012 Apr;1825(2):229-44. doi: 10.1016/j.bbcan.2012.01.004. Epub 2012 Feb 7.
    The p53 circuit board.
  1. Wade M, Wang YV, Wahl GM. 
    Trends Cell Biol. 2010 May;20(5):299-309. doi: 10.1016/j.tcb.2010.01.009. Epub 2010 Feb 19. Review.
    The p53 orchestra: Mdm2 and Mdmx set the tone.
    PMID: 20172729 [PubMed - indexed for MEDLINE] Free PMC Article
  1. Soussi T.
    Hum Mutat. 2014 Jun;35(6):641-2.
    The TP53 gene network in a postgenomic era.
    PMID: 24753184 [PubMed - in process]

Topics: Nano-Traps

News about Nanobodies

For researchers interested in

  • New Nanobody based tools
  • Tips and tricks
  • Featured publications

Recent Posts

Posts by Tag

See all