In this video, I will be showing you how to set up a tissue culture experiment to investigate how treatment of cells with drugs can affect the post-translational modifications of a protein.
You will be analysing the transcription factor Sp3 in brain endothelial cells treated with the drugs verapamil or mithramycin.
The protein under investigation, Sp3, is located in the nucleus, so nuclear extracts are prepared from the cells. You will then be able to perform blots for post-translational modifications - ubiquitin or Sumo, or phosphotyrosine.
However, before you can look at any post-translational modifications, you first need to know how many variants of Sp3 are present in untreated cells. So the first task will be to examine untreated cells and determine the Mr values of Sp3 bands in those cells. As well as staining for Sp3 you will also reprobe the blots for TBP, TATA-binding protein, which serves as an internal standard, to confirm equal loading of your samples.
The tissue culture lab allows you to design many different types of experiment including
… time course experiments, where a single concentration of a drug is used for variable amounts of time
… or dose response experiments, where different drug concentrations are used for a set time.
For this activity, you will carry out a dose response, but you may need to carry out time-course experiments for assessment.
Remember to record the experimental details and results in your notebook.
Now let’s take a look at the tissue culture lab.
________________________________________________________________________________You will find that the tissue culture lab allows you to use different cell types and different drugs but I will just start by showing you how to set up an experiment with brain endothelial cells and mithramycin.
You can see seven tissue-culture plates containing orange tissue culture medium. The first thing is to set the appropriate experimental type by clicking here...to select brain endothelium, ….and here to select the drug – in this case mithramycin.
Each plate can be treated with a different concentration of the drug. Don’t forget that you will need one untreated plate, to see what Sp3 looks like in untreated cells.
I will also select two different drug concentrations; 1 µg ml≄1 on this plate and 5 µg ml≄ on this plate. As this is a dose response experiment, I should set each of the plates for the same incubation time….in this case 12 hours. Every plate needs to be set separately. This allows you to vary the amount of time that the plates are incubated, if you are doing a different type of experiment.
The program allows variable doses up to the maximum concentration of the stock solution, and variable times up to 24 hours. The actual times and concentrations that you will use will depend on your experimental design.
Then the cells are incubated for the set time points by clicking here … while the counter records the passage of time.
When the incubation time on a plate has completed the plate the medium is removed and the cells are harvested.
High drug concentrations, particularly for long time periods, may be toxic for the cells. In this case, the cells will die. They stop releasing carbon dioxide, and the pH of the medium rises, causing it to become red. These plates cannot be used.
If you decide to redo an experiment, press the reset button, here, to clear the old conditions. The nuclear protein isolated from successfully harvested plates is automatically adjusted to 500 µg ml≄, and carried forward in the same order, to the western blotting screen.