Gel Preparation (XCell SureLock Mini-Cell Electrophoresis System, Invitrogen) Separating Gel
Prepare separate gel solution in 50 ml tube (see Table 1&3). Note. TEMED has to be added at the last.
Transfer the gel solution into the cassette using 10 ml pipette tip. Set aside the leftover gel solution.
Layer the top of the gel with 1 ml of 70% EtOH. Note1. Oxygen inhibits the polymerization of acrylamide gel. To make a smooth separating/stacking gel interface, you have to protect the gel solution from oxygen in the air by layering the top of the gel with 70% EtOH. Note2. Ideally, it is required to use isopropanol or butanol. However, 70% EtOH is good enough. It’s cheap and easy to get in every lab. Even H2O can be used but this is not recommended as H2O easily can be mixed with the gel solution thus changing the density of the top of the gel.
Wait for 30 min. Do not sit for over 2 hours.
Check the leftover gel in the tube is polymerized. Go to the next step if the gel is polymerized. Note. If the gel is not polymerized, make the gel again with a freshly made APS solution.
Remove the 70% EtOH and wash out the remaining 70% EtOH. Then, absorb the residual H2O with Kimwipes. Note. Residual EtOH reduces the resolution of protein band.
Table 1. Separating gel recipe.
Stacking Gel
Prepare stacking gel solution in 10 ml tube (see Table 2&3). Note. TEMED has to be added at the last.
Transfer the gel solution on top of separating gel using 10 ml pipette tip. Set aside the leftover gel solution.
Add combs to make wells.
Wait for 30 min.
Check the leftover gel in the tube is polymerized. Go to the next step if the gel is polymerized.
Remove the comb and the tape on the bottom of the cassette. Note. Before removing the comb, mark the position of wells so that you can see the well position easily during the sample loading step.
Table 2. Stacking gel recipe. If the percentage of separating gel is equal to or over 10%, use 5% stacking gel.
Table 3. 4X Separating Buffer and 4X Stacking Buffer recipe.
Table 4. 10X Running Buffer recipe. It must be diluted to 1X running buffer with H2O for SDS-PAGE.
SDS-PAGE
Prepare 500 ml of 1X Running Buffer (50 ml of 10X Running Buffer + 450 ml of H2O)
Pour 500 ml of 1X Running Buffer and run electrophoresis at 80 V through the stacking gel and 150 V through the separating gel until the dye (ladder) has reached the bottom of the separating gel. Note. Voltage can be adjusted. However, too fast speed may cause smear bands.
Role of Compounds
Acrylamide/bis
Acrylamide forms linear polymers and bis-acrylamide forms crosslinks between the linear polymers creating a mesh-like form so that proteins can be separated. A higher percentage of separation gel is required for lower size of proteins whereas larger proteins require lower percentage of separation gel.
25% APS (Ammonium persulfate)
Initiates acrylamide polymer formation.
TEMED
Acts as a catalyst for the acrylamide polymer reaction. Thus, TEMED has to be added at the last.
Tris
Used as a buffering system.
The Principle of Stacking Gel
To get all protein samples lined up so that they can enter the separation gel at the same time. Because of its low percentage, the large pore size of polyacrylamide gel allows the proteins to move more freely so that larger proteins also can move like smaller proteins. On the other hand, low pH (6.8) also helps proteins to equalize with each other. Glycine buffer can exist in two states (zwitterion at lower pH and charged glycinate anion at higher pH). As the pH of stacking gel is low (pH 6.8), glycine moves slow while cl- ions move faster. Therefore, a steep voltage gradient will be developed between Cl- and glycine. And between them, all the proteins exist and stacked into thin bands.
The Principle of Separating Gel
As the pH of separating gel is high (pH 8.8), glycine is ionized. Ionized glycine move faster than proteins. Thus, the steep voltage gradient between the Cl- and Gly- is almost disappeared. Therefore, the mobility of the proteins depends on their mass and the pore size of the gel.
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