I have completed running a number of qRT-PCRs on neuron-like cells transfected with the wild-type version of a transcription factor, a mutant version and an empty-vector. Results are varied, with some genes showing significant differences between the wild type and empty vector but no difference between the wild-type and mutant, while others do show differences between the wild-type and mutant.
I want to find a mechanistic explanation to these results by assessing binding of the mutant and wildtype proteins to the promoter regions of these genes. Whats technique should i use? EMSA (Electrophoretic Mobility Shift Assay) or ChIP (Chromatin Immuno-Precipitation) and why? given that i have a limited time remaining (6-7 weeks)
EMSA or ChIP
1 reply to this topic
Posted 14 December 2010 - 03:56 PM
If you just want to show that your proteins are able to bind to the given sequence, you can do a gel shift. If you want to assess whether binding is actually occurring in the cell, you will have to do ChIP. ChIP does requires a large time investment, whereas the gel shift can be done pretty quickly. You should probably do the gel shift first to determine binding, and if necessary, confirm in vitro using chip. Good luck!