Nov 15, 2019
Polyacrylamide Gel Electrophoresis (SDS-PAGE)
- 1University of Manitoba
Protocol Citation: Neilier Junior 2019. Polyacrylamide Gel Electrophoresis (SDS-PAGE). protocols.io https://dx.doi.org/10.17504/protocols.io.9b7h2rn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 14, 2019
Last Modified: November 15, 2019
Protocol Integer ID: 29791
Abstract
Parameters adjusted for gels 7 cm and 1.5 mm thick.
Materials
MATERIALS
Bromophenol BlueFisher ScientificCatalog #BP11525
Ammonium Persulfate, 25g (Ammonium Persulphate)PromegaCatalog #V3131
SDSBIO-RADCatalog #161-0302
1-ButanolBio Basic Inc.Catalog #BC1800.SIZE.1L
GlycineSigmaCatalog #50046
GlycerolMerck MilliporeCatalog #104092
TEMEDSigma Aldrich
HCl
TrisThermo FisherCatalog #17926
Coomassie Brilliant Blue G-250 DyeThermo FisherCatalog #20279
500 ml 30% acrylamide and bis-acrylamide solution 29:1Catalog ##1610156
Safety warnings
Wear personal protective equipment: gloves, lab coat and mask.
Before start
Organize your workspace
Make sure all solutions and equipment are available.
Preparation of solutions and workspace
Preparation of solutions and workspace
Separation Gel Buffer (150 mL)
1.5 M Tris-HCl, pH 8.8
- Tris (18,15 g/100 mL) ............................................................... 27,23 g
- diH2O ......................................................................................... 80 mL
- Adjust pH to 8.8 with HCl
- diH2O ........................................................................................ to 150 mL
- Store at 4 ° C
Stacking Gel Buffer (100 mL)
0.5 M Tris-HCl, pH 6.8
- Tris ...................................................................................... 6,00 g
- diH2O ................................................................................... 60 mL
- Adjust pH to 6.87 with HCl
- diH2O .................................................................................. to 100 mL
- Store at 4 ° C
10% SDS (100 mL)
• SDS ...................................................................................... 10.00 g
• diH2O ................................................................................... 90 mL
• Solubilize
• diH2O ................................................................................... to 100 mL
10% Ammonium Persulphate (APS) (1 mL)
• APS ................................................................................. 0.10 g
• diH2O .............................................................................. 1 mL
Safety information
Prepare at time of use
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)
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)
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)
Note
These volumes are standardized for making two 7 cm wide and 1.5 mm thick gels.
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)
| Stacking Gel | Separation gel | |||||
| 4% | 7,5% | 10% | 12% | X% | ||
| 30% acrylamide/bis | 1320 µL | 5000 µL | 6666 µL | 8000 µL | 0,666 x X µL | |
| 0,5 M Tris-HCl, pH 6,8 | 2520 µL | - | - | - | - | |
| 1,5 M Tris-HCl, pH 8,8 | - | 5000 µL | 5000 µL | 5000 µL | 5000 µL | |
| 10% SDS | 100 µL | 200 µL | 200 µL | 200 µL | 200 µL | |
| diH2O | 6000 µL | 9700 µL | 8024 µL | 6700 µL | 14,69-(0,666 x X) µL | |
| 10% APS | 50 µL | 100 µL | 100 µL | 100 µL | 100 µL | |
| TEMED | 20 µL | 20 µL | 20 µL | 20 µL | 20 µL | |
| Total volume | 10 mL | 20 mL | 20 mL | 20 mL | 20 mL | |
Safety information
TEMED should only be added just before spilling the solution between plates.
Gel Assembly
Gel Assembly
Clean the workbench, glass plates, spacers, combs and other equipment that will be used with alcohol and assemble the plates
Apply a thin layer of Vaseline to the rubber of the mounting bracket. Check for leaks by adding water. Fully remove water with filter paper
Prepare the separation gel go to step #7 Table 1
Add TEMED
Pour 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.
Safety information
Do not leave n-butanol in contact with gel for more than 1 h
Keep polymerization cassette level
Wait for complete gel polymerization, following what was left of the solution in the erlenmeyer
Remove n-butanol and wash with water
Dry well
Prepare the stacking gel go to step #7 Table 1
Add TEMED and apply the stacking gel to the separation gel (already polymerized)
Immediately put the comb in the gel being careful not to bubbles
Expect full gel polymerization
If necessary, the gel may be stored overnight in running buffer at 5 ° C.
Safety information
Remove the comb only when mounting the running bowl
Sample Preparation
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 microtube
Add 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).
Boil the mix (samples plus sample buffer) for 5 min.
100 °C
Application of Samples
Application of Samples
Attach the plates to the support (gel plate and dead plate).
Place running buffer in the space between the plates (≈ 30 mL).
Check for possible leaks.
Apply the samples in their respective channels.
Add sample buffer to empty channels, if any.
Put running buffer in the bowl halfway.
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.
Follow the race for bromophenol blue.
Stop the race when bromophenol blue reaches the lower limit of the gel.
Safety information
Do not let bromophenol blue escape completely from gel. Low-mass proteins or peptides may be lost.
Remove the gel from the plates.
Mark the gel to reference which side is right and which side is left.
Gel Fixation
Gel Fixation
Place the gel in a fixative solution (10% methanol, 5% acetic acid) and keep stirring for 2 h
Gel Development (coomassie)
Gel Development (coomassie)
Coomassie Staining
Discard the fixative solution
Add 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 mL
Add 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.
Gel Development (silver)
Gel Development (silver)
Silver staining
Discard 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 times
Incubate the gel in silver nitrate solution (0.2 g silver nitrate; 0.037 mL 37% formaldehyde; 100 mL water) for 20 min
Wash the gel for 20 s with water 3 times
Apply 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.