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Thread: HSP70

  1. #1 HSP70 
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    hi there,

    not sure if there's anyone working with hsp70 gene and familiar with its transciption and translation process. i'm working with hsp70 and i found out that there's transcription happened where i detected with pcr method but it was not translated to protein (detection through proteomics approach). anyone familiar with untranslated region in mrna or can explain how this thing can happen? is it solely because of the untranslated region in mrna?

    thanks and please advice...

    regards
    kenny


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      Him
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      Transcription and translation rates often do not match each other. In your case this is a pity hence the reviewers want to see the proteins changing if you confirm real time PCR results. The other way around is more acceptable.
      Not only the difference in translation and transcription account for differences between mRNA and protein content also alternative splicing and post translational modification have to be regarded. About 40-60% of the human genes have alternative splicing. 2-dimensional gel electrophoris and Mass spectrometry studies demonstrate that transcription of one gene results in an average of 10-15 different protein spot (different spliced, modified and isoforms).

      Here are some references:

      Anderson L, Seilhamer J. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis. 1997;18:533-537.
      Fountoulakis M, Juranville JF, Berndt P, Langen H, Suter L. Two-dimensional database of mouse liver proteins. An update. Electrophoresis. 2001;22:1747-1763.
      good review regarding difference transcription-translation: Two-dimensional gel electrophoresis; better than a poke in the ICAT? Curr Opin Biotechnol. 2002;13:321-328.

      Most of the reasons I have given are used to explain when one sees the protein changing in a proteomic setup and can not confirm this by real time PCR.
      Maybe another thing since you say you can’t detect any HSP70 is that pcr is much more sensitive then the proteomic approach. On the other hand to my knowledge HSP70 is a abundant protein so you should be able to detect it (but all dependent on the system you are working in of course).

      If you can detect HSP70 I would suggest that you do a two dimensional separation of your extract and perform a western blotting on this. (first optimize your blotting on 1 dimensional separation of course). If then you do not see any changes of certain HSP70 isoforms then there is nothing going on (only a higher transcription, compensated by a higher turnover of the mRNA molecule; but to assure you this will not be the case I am convinced you’ll see some differences when performing the 2D-blotting).
      Can you tell me what your proteomic approach was? this can help...


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