TITLE: Polyacrylamide Gel Electrophoresis (SDS-PAGE)
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Parameters adjusted for gels 7 cm and 1.5 mm thick.</div></div>

[STEPS]
?. [Preparation of solutions and workspace]
Separation Gel Buffer (150 mL)1.5 M Tris-HCl, pH 8.8Tris (18,15 g/100 mL) ............................................................... 27,23 gdiH2O ......................................................................................... 80 mLAdjust pH to 8.8 with HCldiH2O ........................................................................................ to 150 mLStore at 4 ° C
?. [Preparation of solutions and workspace]
Stacking Gel Buffer (100 mL)0.5 M Tris-HCl, pH 6.8Tris  ...................................................................................... 6,00 gdiH2O ................................................................................... 60 mLAdjust pH to 6.87 with HCldiH2O .................................................................................. to 100 mLStore at 4 ° C
?. [Preparation of solutions and workspace]
10% SDS (100 mL)• SDS ...................................................................................... 10.00 g• diH2O ................................................................................... 90 mL• Solubilize• diH2O ................................................................................... to 100 mL
?. [Preparation of solutions and workspace]
10% Ammonium Persulphate (APS) (1 mL)• APS ................................................................................. 0.10 g• diH2O .............................................................................. 1 mL
Prepare at time of use
?. [Preparation of solutions and workspace]
10x Running Buffer (1 L)• Tris base ...................................................................... 30.30 g• Glycine ......................................................................... 144.10 g• SDS ............................................................................... 10.00 g• diH2O ............................................................................ for 1 L• Do not adjust pH (~ pH 8.3)
?. [Preparation of solutions and workspace]
Sample Buffer (8 mL)2X: 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue• 0.5 mM Tris-HCl, pH 6.8 ………………………...……….. 1.0 mL• 25% glycerol ………………………………………………………. 2.0 mL• 1.0% bromophenol blue ……………………....…………. 0.08 mL• 10% SDS …………………………………………………......……. 1.6 mL• diH2O ………………………………………………………......…… 2.92 mL
?. [Preparation of Denaturing Gels (SDS-PAGE)]
Choose which concentration of separation gel to use and separate two erlenmeyer (one for separation gel and one for stacking gel)Table 1. Preparation of 20 mL SDS-PAGE Running Gel (2 x 1.5 mm gels) and 10 mL Stacking Gel (2 x 1.5 mm gels)  ABCDEF1     Stacking GelSeparation gel24%7,5%10%12%X%330% acrylamide/bis  1320 µL    5000 µL    6666 µL    8000 µL    0,666 x X µL  4  0,5 M Tris-HCl, pH 6,8    2520 µL    -    -    -    -  5  1,5 M Tris-HCl, pH 8,8    -    5000 µL    5000 µL    5000 µL    5000 µL  6  10% SDS    100 µL    200 µL    200 µL    200 µL    200 µL  7diH2O  6000 µL    9700 µL    8024 µL    6700 µL    14,69-(0,666 x X) µL  8  10% APS    50 µL    100 µL    100 µL    100 µL    100 µL  9  TEMED  20 µL20 µL20 µL20 µL20 µL10Total volume  10 mL    20 mL    20 mL    20 mL    20 mL
These volumes are standardized for making two 7 cm wide and 1.5 mm thick gels.
ABCDEF1     Stacking GelSeparation gel24%7,5%10%12%X%330% acrylamide/bis  1320 µL    5000 µL    6666 µL    8000 µL    0,666 x X µL  4  0,5 M Tris-HCl, pH 6,8    2520 µL    -    -    -    -  5  1,5 M Tris-HCl, pH 8,8    -    5000 µL    5000 µL    5000 µL    5000 µL  6  10% SDS    100 µL    200 µL    200 µL    200 µL    200 µL  7diH2O  6000 µL    9700 µL    8024 µL    6700 µL    14,69-(0,666 x X) µL  8  10% APS    50 µL    100 µL    100 µL    100 µL    100 µL  9  TEMED  20 µL20 µL20 µL20 µL20 µL10Total volume  10 mL    20 mL    20 mL    20 mL    20 mL
TEMED should only be added just before spilling the solution between plates.
?. [Gel Assembly]
Clean the workbench, glass plates, spacers, combs and other equipment that will be used with alcohol and assemble the platesApply a thin layer of Vaseline to the rubber of the mounting bracket. Check for leaks by adding water. Fully remove water with filter paper
?. [Gel Assembly]
Prepare the separation gel Add TEMEDPour the separating gel solution onto the plate up to the demarcated limit for the stacking gel.Apply a thin layer of n-butanol over the separation gel to level the upper portion of the gel.Wait for complete gel polymerization, following what was left of the solution in the erlenmeyer
[Table 1]
Do not leave n-butanol in contact with gel for more than 1 hKeep polymerization cassette level
?. [Gel Assembly]
Remove n-butanol and wash with waterDry well
?. [Gel Assembly]
Prepare the stacking gel Add TEMED and apply the stacking gel to the separation gel (already polymerized)Immediately put the comb in the gel being careful not to bubblesExpect full gel polymerizationIf necessary, the gel may be stored overnight in running buffer at 5 ° C.
[Table 1]
Remove the comb only when mounting the running bowl
?. [Sample Preparation]
Calculate the sample volume required to provide the desired amount of protein (≈ 30 µg / channel)Distribute the calculated sample volume into 600 µL microtubeAdd 2X sample buffer to samples at a 1:1 ratio.If sample buffer 4X, use 3:1 ratio (in this case apply for each channel 5 µL of sample buffer, add sample and complete with diH2O to 30 µL on 1.5 mm gel).
?. [Sample Preparation]
Boil the mix (samples plus sample buffer) for 5 min.
100 °C
?. [Application of Samples]
Attach the plates to the support (gel plate and dead plate).
?. [Application of Samples]
Place running buffer in the space between the plates (≈ 30 mL).Check for possible leaks.
?. [Application of Samples]
Apply the samples in their respective channels.Add sample buffer to empty channels, if any.
?. [Application of Samples]
Put running buffer in the bowl halfway.
?. [Application of Samples]
Fit the electrodes and turn on the device for running, using 200 V.Expected current of gel: initial 35 - 50 mA and final 20 - 31 mA.
?. [Application of Samples]
Follow the race for bromophenol blue.Stop the race when bromophenol blue reaches the lower limit of the gel.
Do not let bromophenol blue escape completely from gel. Low-mass proteins or peptides may be lost.
?. [Application of Samples]
Remove the gel from the plates.Mark the gel to reference which side is right and which side is left.
?. [Gel Fixation]
Place the gel in a fixative solution (10% methanol, 5% acetic acid) and keep stirring for 2 h
?. [Gel Development (silver)]
Silver stainingDiscard the fixative solution and perform three washes of 10 min with 50% ethanol (92% commercial ethanol may be used in this step)Incubate the gel with 0,02% sodium thiosulphate solution for 1 min.Wash the gel in distilled water 3 timesIncubate the gel in silver nitrate solution (0.2 g silver nitrate; 0.037 mL 37% formaldehyde; 100 mL water) for 20 minWash the gel for 20 s with water 3 timesApply developer solution (2 g of sodium carbonate; 1 mL of 0.02% sodium thiosulfate solution; 0.025 mL of 37% formaldehyde; complete with water to 50 mL)Wait for the bands to appear in a few seconds. In some cases, it takes a few minutes for the bands to become evident.Once the gel reaches the required standard, stop the reaction by adding 3 mL of acetic acid.
?. [Gel Development (coomassie)]
Coomassie StainingDiscard the fixative solutionAdd Coomassie's solution, which is:• Ammonium sulfate (10%): 100 g• Coomasie G250 (0.1%): 1 g• Water: 800 mL• Phosphoric acid (2%): 20 mL• Methanol (20%): 200 mLAdd ammonium sulfate and Coomassie G250 to 400 mL of water until the sulfate dissolves.Add phosphoric acid, followed by methanol slowly. Finally, add the other 400 mL of water.