This video will demonstrate how to perform SDS-PAGE for protein characterization.
SDS-PAGE stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Here is the equipment you will need.
First, insert a short plate and a 1 mm spacer plate into the casting frame.
Make sure the feet of the casting frame are on the bottom.
Make sure the plates are also levelled at the bottom.
Then tighten the clamp to secure the position of the plates.
Next, install the casting frame with the plates onto the casting stand
and ensure the setup is tight and secured.
Check the seal of your set-up by pipetting dd water halfway up the plates.
If there are no leaks, then remove the ddwater either using kimwipes or filter paper,
until it is almost dry.
When the gel casting stand is set up, prepare the 12% resolving gel solution in a 50 mL falcon tube.
Mix 3.3 mL of dd water,
4.0 mL of 30% acrylamide solution
Liquid Acrylamide is neurotoxic and should be handled with caution.
2.5 ml of lower stock,
and 100 microliters of 10% SDS together.
Right before you are ready to pour the gel, add 10 microliters of 10% APS
and 4 microliters of TEMED.
Please note that TEMED has to be added in the fume hood due to its strong odour.
TEMED catalyzes the polymerization of acrylamide.
If TEMED is added way too early , the gel will set before it is ready to be pipetted in between the plates.
Mix gently, then immediately pipette the resolving gel solution in between the plates
with a glass pipette, until the resolving gel reaches just above the green line,
or approximately 1cm from the top of the short plate.
Please monitor to make sure there is no leak at the bottom of the plates.
Pipette 200 microlitres of isopropanol on the resolving gel.
You should observe a thin layer on the gel.
While waiting for the resolving gel to polymerize,
prepare the stacking gel solution in another 50 mL falcon tube.
Mix 1.35 mL dd water, 0.335 mL acrylamide stock, 0.25 mL upper stock and 20 microliters of 10% SDS.
Once the resolving gel has formed which takes roughly 30 minutes , drain the isopropanol.
Gently rinse the top of the gel with dd water and dry with kimwipes.
Right before you are ready to pour the stacking gel,
add 20 microliters of 10% APS and 2.0 microliters of TEMED into the stacking gel solution, mix well.
Immediately pipette about 1 mL of the stacking gel solution on top of the resolving gel in between the plates.
Fill to the top, then insert the comb into the solution.
Wait for the gel to polymerize.
After the gel is formed, unclip the casting frame with the gels from the casting stand,
then carefully remove the glass plates from the casting frame.
Wrap the plates carefully in wet paper towel and saran wrap,
then store in the cold room until next week.
To prepare samples for SDS-PAGE, label the microfuge tubes corresponding to your samples.
Pipette 40 microlitres of the fish extract into the microfuge tube
and add 40 microliters of sample buffer.
Heat the tubes at 100 degrees Celsius for 3 minutes.
Then pulse centrifuge for 5 seconds.
Remove the gel from the saran wrap and paper towel.
Tightly secure the gel with the short plate facing inwards, onto the running module.
Each running module can hold 2 gels.
If only 1 gel is being run, you will need to use the buffer dam in place of the second gel.
Do not directly label your plates with tape because this will prevent the gel from running properly.
Instead, plates can be directly labelled with a permanent marker.
Each tank can accommodate two running modules, which will be 4 gels altogether.
Insert the running module into the tank, make sure the module properly inserts into the slot in the tank.
At this point, check to see if the lid fits appropriately.
Pour the 10x diluted running buffer solution into the running module between the two plates
until wells on the two plates are fully submerged.
Check to make sure the running modules aren’t leaking any buffer solution into the tank.
The tank only needs to be filled half way with the buffer solution if running 2 gels.
Now you are ready to load the samples.
Remove the comb so you can see each well clearly.
You can install the loading guide on the gel to make it easier to load your samples properly into each lane.
Carefully load 10 microliters of sample solution into each well.
The pipette tip should be inserted into the well in between the plates,
deep enough to prevent overflow of the sample to the neighbouring lanes.
Slowly release the sample solution into the lane.
When complete, remove the pipette tip, change the tip, then load another sample.
For the molecular weight marker, you also need to load 10 microlitres into the well
and it should be loaded in one of the center lanes for easy comparison.
When all the samples are loaded, close the lid on the tank
and insert the electrodes properly into the power supply,
red wire into the red slot and black into the black slot.
Set the power to 20 milliamps per plate and run for 1 hour.
For example, if 4 plates are being run, the power should be set at 80 milliamps.
The power can also be set to voltage.
The voltage used isn’t dependent on the number of plates
so a consistent voltage can be used to run any number of plates.
For example, a consistent 120 volts can be used.
You should initially observe the sample or standard solution to be condensed into a thin band through the stacking gel,
then the band will gradually migrate down the resolving gel.
When the blue dyes have reached close to the bottom of the gel, stop the run.
Unplug the power supply, then remove the running module from the tank and drain the running buffer.
Remove the glass plates carefully from the running module,
then carefully use the gel releaser to remove the gel from the glass plates.
You can trim off the stacking gel carefully before proceeding to staining.
Transfer the gel into a square petri dish.
Then pour the Coomassie blue staining solution into the petri dish, submerging the gel fully.
Place the square petri dish in a metal tray to avoid spillage in the incubator
then place in a 50°C incubator to stain for 30 min.
Remove the gel with a tweezer and make sure you can observe clear bands on the gels,
before discarding the staining solution and replacing it with the destaining solution.
When destaining, add a piece of Kimwipe or absorbent paper towel to help absorb the dye.
Destain at 50°C for 30 minutes, with periodic swirling to facilitate the destaining process.
You may also want to change the destaining solution every 15 minutes,
until the background of the gel is transparent.
When the destaining is complete, store the gel in dd water
until you can scan and analyze it with the densitometer
Properly dispose of any liquid acrylamide-containing solutions in the acrylamide waste container,
Polymerized gels can be disposed directly in the garbage,
while the staining and destaining solutions can be disposed into the non-halogenated waste container.