3 Consider a cotransfection assay with expression vectors fo

3 Consider a cotransfection assay with expression vectors for two putative transcription factors that you suspect work together to activate transcription, and with a regulatory region of interest attached to a CAT reporter gene. You also test two deletion (A) mutants of the regulatory region. (a) How could you confirm that the transcription factors are transcribed and translated in the transfected cells? (b) Interpret the results of the CAT assay below. The plus and minus symbols indicate the presence or absence of expression vectors for the listed components in the transfection a Transcription factor A Transcription factor B Full-length regulatory region (30 bp) Deletion mutant l (AI-15) Deletion mutant 2 (A16-30) (c) How would you demonstrate protein-protein interaction between the two transcription factors in vivo and in vitro? Show sample positive results. (d) Describe protocols to further characterize direct binding of the proteins to DNA in vitro and in vivo. Show sample results

Solution

(a) for checking whether the the translation is performed we can perform a sds page of the transfected cell and can check for the desired protein bands in the gel..!!

(c) for checking the protein - protein interactions in-vivo we can use a technique called FRET. in which through flouresnce we can detect the protein interaction as one of the protein is flouresnce labeled and through excitation the other proteins of interest will flourscene at a certain desired wavelength..

In - vitro as the protein will binds to each other hence the size will increase which can be checked by the native page which will give the single proteins bands in desired size after combining..

(d) In-Vitro we can determine the interaction by X-ray crystallography where we can easily detect the atomic structure of the DNA.

In-vivo one can determine by Chromatin immuno precipitation assay.