Protocol for Preparing SDS-PAGE Gels
Please click this hyperlink to access the virtual lab bench
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Materials and Reagents
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Protocol for preparing and loading protein samples
Protein molecular weight marker (only one marker lane/gel)
- One sample of the cell lysate collected
- One sample of the flow-through
- Wash fraction 1 sample
- Wash fraction 2 sample
- Elution fraction 1 sample
- Elution fraction 2 sample
- Elution fraction 3 sample
Prior to the lab, please use your Bradford assay data and calculate the amount (in µg) of protein you will be loading in each of your elution fraction samples.
2. You will be given a Tupperware container to store your gels in. It may be necessary for more than one student to share a Tupperware container. This means that you need to be able to tell your gel apart from the other gels in the container. The best way to do this is to move the marker lane to different spots on the gel for each lab bench. So, if you have a 15-lane gel, you can claim which lane on the gel your marker will reside in. Please keep in mind that the gels are transparent and there is no way of telling if it is flipped. What do we mean by this? Let’s look at all possible combinations for placing the marker (which will look like multiple blue bands so really easy to distinguish) in each lane:
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3. Using 1.5 mL Eppendorf tubes, aliquot 15 µL of the above samples and 15 µL of 2X SDS-PAGE loading buffer into each tube. When you are done return your protein fractions to the -20 °C freezer.
4. Boil your samples for 2-3 minutes. Take care when adding and removing samples from the hot plate. The hot plate is HOT which means that the sample tubes are hot so please use the tweezers provided to handle the tubes.
5. Give your tubes a quick spin (~1-2 sec) in a micro-centrifuge to spin down any condensation that may have formed during boiling.
6. Your instructor will show you how to load the wells. You should load 15 µL of sample/well. For your marker lane, load 5 µL of prestained protein ladder. Let your instructor know when you are ready to run your gels (note that two gels are run in one electrophoresis tank).
Protocol for gel electrophoresis
Your instructor will help everyone get their gels ready to run by setting up the electrophoresis tank. Please pay attention: they will go over the basics.
The electrodes will be connected: (-) ve black, (+) ve red. Turn on and adjust the voltage to 170 volts (this is the maximum voltage). The run time for the gels is approximately 45 minutes. Please be careful as this is a high voltage instrument. Do NOT touch the instrument while it is running, do not run the instrument in the presence of water on the bench top.
- When the dye front is at the very bottom of the separating gel (90 % migration), YOUR instructor WILL turn the voltage to zero and then turn the power supply off and unplug the instrument. Remove the electrode assembly, unclamp the assembly and gently remove the gel sandwich. Using a gel releaser (green instrument provided), gently break the seal between the two plates and remove the gel (the stacking gel can be discarded in a paper towel and placed in the biohazardous waste). Your mentor will help you with these steps. Caution must be exercised because polyacrylamide gels are fragile.
- Place your gels in water (within a sandwich-sized Tupperware). Your gels will be stained, dried and a picture of the gel will be posted on the course learning management system sometime during the following day .
Please click this hyperlink to see what the gel looks like
Once you are finished please wash your hands!
