Experiment 1. Optimising resolution and detection of ABCB1 and determining its relative molecular mass
Materials
· Caco-2 cell lysate (total protein concentration = 1.5 mgml−1).
· Sample diluent
· Three detection antibodies (each at a suitable concentration for use; in the range 0.1–2.0µgml−1)
· Rainbow markers 225000 blue, 150000 red, 98000 green, 68000 yellow, 45000 purple, 36000 orange, 24000 blue, 17000 red.
Methods
In the virtual laboratory you will be able to change a number of parameters:
· the concentration of acrylamide in the gel
· the amount of protein which is loaded on each lane of the gel
· the choice of primary antibody used to detect ABCB1.
[The protocol below lists all the steps needed to run this experiment. The key steps which you carry out in the onscreen laboratory are in bold.]
Protocol
1 Prepare a range of dilutions of the Caco-2 cell lysate using the sample diluent. Each sample should have a final volume of 100 ml. Prepare at least five samples in the range
20–1000µgml−1 and a negative sample (no protein).
2 Add 20 ml reducing sample buffer to each sample and heat at 95°C for 5 min.
3 Prepare an acrylamide gel, selecting a suitable acrylamide concentration to resolve ABCB1.
4 Load 20 ml of each sample onto separate lanes of the gel and markers onto the final lane.
5 Run the gel for 45 minutes at 100 V and then blot the gel. [Note that when you run your gel in the virtual laboratory, you will only see the rainbow markers. The bands will become visible when you have added your antibody and visualised your blot.]
6 Detect ABCB1 by incubating the blot with each of the three available primary antibodies [Incubate in 2 ml primary antibody for 1 h, followed by washing (x3), incubation in 2 ml 0.3 µgml−1 peroxidase-conjugated anti-mouse-IgG (secondary antibody) for 1 h and further washes (x3)].
7 Visualise the blots by chemiluminescence.
Take screen shots of your blots, incorporate them with these notes in your experimental notebook and use one blot to determine the Mr of ABCB1.