The transporter protein ABCB1 prevents many therapeutic drugs from entering the cell, and consequently is of great importance for the pharmaceutical industry. At the molecular level, the expression of ABCB1 is controlled by a number of transcription factors, including Sp1 and Sp3. In this investigation you will use the Western blotting technique to characterise ABCB1 and Sp3 and to analyse their expression in a number of different cell types.
First, you will learn more about the following techniques which underpin the experiments:
SDS–PAGE and Western blotting
interpretation of gels and blots
relative mobility calculations and Mr determination
quantitation of signals in Western blots.
The investigation consists of three laboratory experiments with each experiment building upon the previous one, so you should go through them in order. In each investigation you will be presented with a series of specific aims and guidance on how to carry out the experiment.
As well as helping you understand aspects of RNA-splicing and post-translational modification of proteins, this Western blotting investigation will help you to develop a number of key practical skills:
manipulation of concentrations, volumes and dilutions
calculation of the relative molecular mass (Mr) of proteins based on relative migration
optimisation of experimental conditions, to obtain meaningful data
using band density measurements to determine relative protein abundance in samples analysed by Western blot
experimental design and interpretation of results
report writing.
This is a substantial activity and it is unlikely that you will complete it in one sitting. You can stop and return to the activity at any time. Remember to save your work.
Do not be tempted to skip the background and theory content and practice activities, as this may affect your ability to conduct the experiments as required and to interpret your findings. To check your understanding, you should undertake the practice activities and complete the quiz questions that are included. While you should work through the Investigation from start to finish, you may wish to return to some of the background and theory section at particular points when you are planning, conducting and interpreting the results from your experiments.
You are strongly advised to keep a hard copy or electronic experimental notebook to record what you do and the results that you obtain in the course of this investigation. The protocols provided in this investigation can be pasted into your experimental notebook and then modified as required. You should save any results from your experiments (for example, blots or densitometry readings) as you work. You should also record any calculations that you perform and any observations that you make in the course of your investigation.
SDS–PAGE is a standard technique for determining the molecular size of a protein and its polypeptide chain composition. It is also very useful for separating complex mixtures of proteins, and gives a good impression of the relative abundance of each protein.
Watch Slidecast 1 and read the associated notes which together explain the theory and principles of the SDS–PAGE and Western blotting techniques.
Slidecast 1 Principles of the SDS–PAGE and Western blotting techniques.
Video 1 shows the SDS–PAGE and Western blotting techniques being performed in a laboratory. You should also read the document Notes to accompany video demonstration of SDS–PAGE and Western blotting, to help your understanding:
Video 1 Demonstration of SDS–PAGE and Western blotting.
Work through this quiz to check your understanding of the theory of SDS–PAGE and Western blotting.
Placeholder for ITQ
If you have problems completing the quiz questions, you should re-read and ensure you fully understand the background theory and watch the videos again.
You have now completed the ‘Introduction to SDS–PAGE and Western blotting’. Return to the Analysing gene expression – Western Blotting landing page to begin the next section ‘Preparing for your experiments’.