In this SDS PAGE principle and Protocol article we will discuss about SDS PAGE protocol for protein sample preparation, gel casting, running the sample and visualization of SDS PAGE protocol for protein.
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis, or SDS PAGE protocol for protein separation depending on their molecular weight. It is a technique for separating protein molecules based on their electrophoretic mobility that is frequently used in forensics, genetics, biotechnology, and molecular biology.
SDS PAGE principle and Protocol
SDS PAGE protocol for protein based principle that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation of the charged molecules depends upon the relative mobility of charged species.
The smaller molecules migrate faster due to less resistance during electrophoresis. The structure and the charge of the proteins also influence the rate of migration. Sodium dodecyl sulphate and polyacrylamide eliminate the influence of structure and charge of the proteins, and the proteins are separated based on the length of the polypeptide chain.
In the SDS PAGE protocol for protein sample buffer, SDS is a detergent. SDS, along with some reducing chemicals, disrupts the tertiary structure of proteins by breaking their disulphide linkages. The degree of SDS PAGE protocol for protein binding is very high, and thus the charge on the SDS PAGE protocol for protein complex is almost entirely due to the exposed sulfate ions.
1. 40% Acrylamide: Acrylamide 116.8 g, N,N’-Methylene bisacrylamide 3.2 g, DD H2O to 300 ml.
2. 30% Ammonium Persulfate: Ammonium Persulfate 1.5 g, DD H2O 5ml (Store at 4°C. Replace every month).
3. 1.5 M Tris HCl, pH 8.8: DD H2O 300 ml, Tris–free base 90.75 g, Conc. HCl 8 ml (Adjust to pH 8.8 with conc. HCl, and bring final volume to 500 ml with DD H2O).
4. 1.0 M Tris HCl, pH 6.8: DD H2O 300 ml, Tris–free base 60.54 g Conc. HCl 36 ml (Adjust to pH 6.8 with conc. HCl, and bring final volume to 500 ml with DD H2O).
5. 4x SDS-PAGE Sample Buffer: 125 mM Tris HCl, pH 6.8 1 M 5 ml, 20% Glycerol 8 ml, 4% SDS 20% 8 ml, 10% ß-Mercaptoethanol 4 ml, 0.5 mg/ml Bromophenol Blue 20 mg, DD H2O 15 ml.
6. 10x SDS-PAGE Running Buffer: 30.3 g Tris base, 144.0 g Glycine, 10.0 g SDS (Dissolve and bring total volume to 1,000 ml with deionized water. Do not adjust pH with acid or base (pH is normally 8.3 as prepared).
7. Coomassie Stain Solution: Ethanol 150 ml, Glacial Acetic Acid 50 ml, DD H2O 300 ml, Coomassie Brilliant Blue–R-250 1 g (Dissolve Coomasie Brilliant Blue–R-250 in EtOH first).
8. Destain Solution: Ethanol 1200 ml, Glacial Acetic Acid 400 ml, DD H2O 2.4 l ((pH is neutral as prepared).
Preparing Samples for SDS-page for protein separation
1. Cell Samples
Harvest 100 µl of cells with an O.D. greater than 0.6. Remove the supernatant medium and decant it. Cells should be resuspended in 20 µl of 2x sample buffer. Incubate tubes for 5 minutes in boiling water. Centrifuge for 30 seconds at 12,000 x g.
2. Solution Samples
Fill a microfuge tube halfway with protein solution (or 1 µl of standard) until the solution contains 5-10 g of protein. 2x sample buffer (or 10 µl for standards) in an equivalent volume Incubate tubes for 5 minutes in boiling water. Centrifuge for 30 seconds at 12,000 x g.
SDS page protocol for protein
Casting the Gel
In a SDS PAGE protocol for protein gel casting device, assemble glass plates and spacers. As needed, combine the components for the resolving gel. Fill the gel plates with the resolving gel mixture to a level 2 cm below the top of the shorter plate.
On prevent meniscus formation in the resolving gel, apply a layer of DD H2O to the top of the gel. Allow 30 minutes for the resolving gel to sit at room temperature. Drain the DD H2O that has accumulated on top of the resolving gel. Drain and wick any residual DD H2O away with a wipe after rinsing with DD H2O.
To make the stacking gel, combine all of the ingredients in a mixing bowl. Stacking gel solution (on top of running gel) should be poured onto gel plates until they are completely filled. Place the comb on top of the spacers. Allow gel to sit at room temperature for at least 1 hour, or overnight at 4°C.
Running the Gel
Remove the comb from the cast gel and place it in the SDS PAGE protocol for protein electrophoresis device. Fill both chambers of the apparatus with freshly made 1x running buffer (300 ml). Place the prepared samples in the gel’s wells.
Run the gel at 100 V for 15 minutes or until the dye front migrates into the running gel, then raise to 200 V for 45 minutes or until the dye front hits the bottom of the gel.
Staining & Destaining the Gel
Remove the spacers and glass plates from the equipment as well as the run gel. Fill a small tray with the gel.
Stain for > 30 minutes with gentle shaking and a 20 ml staining solution. Pour off the liquid and save the stain. Destain for 1 minute with careful shaking after adding 5 mL destain solution.
Pour off and discard the destain solution. Add ~ 30 ml of destain solution. Destain with gentle shaking until the gel is visibly destained (> 2 hr). Pour off and discard the destain solution.
Rinse with DD H2O. Add ~30 ml DD H2O and rinse for 5 min with gentle shaking. Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper below the gel and a piece of Seran wrap over the gel.