TITLE: SPS-PAGE protein electrophoresis
AUTHORS: Konstantinos Ragios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a variant of PAGE that allows to separate protein molecules according to their molecular masses under electric field.</div><div class = "text-block">SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 KDa.</div><div class = "text-block">In this protocol we will use an already precasted gel.</div></div>

[STEPS]
?. [Sample preparation]
In a tube add 10μl of 2x Laemmli Buffer , 5μl Nuclease free water and 5μl of your protein sample.
You may adjust the ratio of water:protein depending on the concentration of proteins in your sample.e.g. for high concentration add 7μl of water and 3μl of proteins        for low concentration add 3μl of water and 7μl of proteins
?. [Sample preparation]
Mix well, either pipetting  up and down, vortex or flicking the tube. Afterwards, a few seconds of centrifuge might be needed to collect all the sample back at the bottom of the tube.
?. [Sample preparation]
Incubate the tube at 100oC for 15 minutes in a dry heating element.
For PCR tubes you may use a Thermal Cycler. Do not forget to set Heated Cover Temperature to 105oC to avoid condensation.
After 15min you can store the samples at 4oC until you start the electrophoresis
?. [Electrophoresis system preparation]
Rinse the tank, the buffer dam (if you want to run an odd number of gels) and the casting stand.
?. [Electrophoresis system preparation]
Open the casting gel, remove the comb and the plastic cover on the bottom and rinse well.
?. [Electrophoresis system preparation]
Insert the gel(s) in the casting stand(s). If you want to run one or three gels you must include a buffer dam to complete the set.
Add some water inside the casting stand between the two gels. If there is no leakage then the gels are mounted properly. Discard the water.
?. [Sample preparation]
Mix 10μl of 2x Laemmli Buffer with 10μl Nuclease free water to use as a negative control.
?. [Electrophoresis system preparation]
Put the casting stand in the tank and fill the inner compartment with 1x TGS Buffer. Then fill the tank until the appropriate marked line.
For one or two gels until the 2 Gels mark, for three or four gels until the 4 Gels mark
?. [Loading the samples]
It is better to use long tips. 20μl tips can also be used but it requires more effort to load the samples without damaging the gel.
?. [Loading the samples]
For the ladder load 3μl in one well, while for the samples and the negative control load 10μl per well.
To make sure that the tip is actually inside the well move it back and front. If your movement is not constricted by the glass frames this means that you are not in the well. Pipette slowly to make sure that no sample will be propelled outside the well.
?. [SDS-PAGE electrophoresis]
Close the lid and connect the electrodes on the power supply.
Make sure that the electrodes are attached to the right position (red in red and black in black)
?. [SDS-PAGE electrophoresis]
Set the power supply to 120Volts for 5 minutes.
After a while you should be able to see some bubbles, if not, then there is a problem with the circuit. Check the cables and add some more buffer in the tank and between the gels
?. [SDS-PAGE electrophoresis]
Then set the power supply to 200Volts and run for 25 minutes
By the end of the 25 minutes the blue band that appears on the gels should be close to the bottom.You may run the gel for a couple more minutes at 200volts so the band will be even closer to the end (to achieve better separation) but be careful not to surpass it.
?. [Gel staining]
Take the casting stand of the tank and pour the buffer in a bottle along with the buffer inside the tank. It can be used for multiple gel runs.
?. [Gel staining]
Carefully open the glass frames. Use the specialized tool or a metal object that can fit in the frame openings.
Every time you touch the gel use clean gloves to avoid contamination.
Leave the gel attached to one of the glass frames so you can transfer it easily.
?. [Gel staining]
Clean the gel.
?. [Gel staining]
Pour distilled water in a bowl.Then put the gel in the bowl.
Keep in mind that in a following step we will put the bowl in a shaker, so be careful not to overflow it.
You can flip the frame in the water an shake gently. Water will come between the gel and the frame so it will be released without any damages
?. [Gel staining]
Put it on a shaker for 5 minutes.
?. [Gel staining]
Remove the water, then add some more clean one and shake for another 5 minutes
Press the gel to the bottom of the bowl using your fingers to avoid risking it falling in the sink
?. [Gel staining]
Repeat step 14.3 one more time
?. [Gel staining]
Remove the water from the bowl and add around 50ml of Bio-Safe Coomassie Stain
Depending on the size of your bowl , you might need some more stain so the gel is completely covered.
?. [Gel staining]
Put on the shaker for one hour.
?. [Gel Washing]
Remove the Coomassie stain and rinse the gel very well
?. [Gel Washing]
Add some distilled water on the shaker and let it overnight (12-16 hours)
You may add some precision wipes in the bowl that will absorb some of the color. Try not to put them on top of the gel because they might be attached to it and when you remove them it will damage the gel
?. [Gel Washing]
Optionally after some hours you can renew the water in the bowl and change the wipes.
Do not overexpose the gel to water because after some time the desired bands might also start to fade away.
?. [Visualization of results]
Carefully remove the gel from the bowl and put it in a transparent colorless plastic file folder.
?. [Visualization of results]
The image of the gel can be captured using a conventional scanner or with the white light mode on a UV transilluminator