You are now asked to carry out an experiment to investigate ubiquitination of Sp3 isoforms in Caco-2 cells and to establish the effect of different concentrations of verapamil treatment at a single time point – i.e. to establish a dose response for the drug.
Note that, with longer exposure times and higher doses, verapamil treatment may kill the cells. You will therefore need to first carry out a pilot experiment to identify a suitable incubation time and dose range for the verapamil treatments to establish a full dose response, whilst ensuring that all of the cultures may be harvested successfully (i.e. the cells have survived)
Your investigation should allow you to make some observations about whether and to what extent Sp3 isoforms are ubiquitinated in Caco-2 cells under basal conditions (without treatment) and in response to verapamil, and to establish the dose-dependency of any effects of verapamil.
Important notes:
You are not required to determine the Mr values for any Sp3 bands, nor are you required to make densitometry readings of bands. However, from visual inspection of your blots you should be able to make some observations about whether, and to what extent, the different Sp3 isoforms are ubiquitinated in response to verapamil in Caco-2 cells.
To help you interpret your observations, you should note that untreated Caco-2 cells express four Sp3 isoforms (with Mr values of 119000, 116 000, 96 000 and 93000)
You should produce a laboratory report on your experiment, structured as follows:
an Introduction in which the aim of your experiment is clearly stated
a Methods section which includes details of what you did and the experimental conditions
a Results section that includes: annotated pictures of no more than two Western blots that provide evidence to support your conclusions, a description of the results from your experiment and your observations.
[Note that screenshots should be cropped appropriately to show the blots; it is not acceptable to screenshot the whole experimental window.]
a Conclusion.