Objective:
To separate proteins on the basis of their size and charge.
Theory
PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size. To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. For this different protein molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or quaternary structure .The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder(marker).
Structure of SDS
Principle behind separation:
Separation of charged molecules in an electric field is based on the relative mobility of charged species which is related to frictional resistance.
Charge of the species:
PAGE is working upon the principle in which, the charged molecule will migrate towards the oppositive charged electrode through highly cross linked matrix. Separation occurs due to different rates of migration occurs by the magnitude of charge and frictional resistance related to the size.
Relative Mobility:
where,
Z = charge on the molecule
E = Voltage applied
and ,
f = frictional resistance
Rf is measured by:
Direction of movement is determined from Z: -
if Z < 0, then →+
if Z > 0, then → -
if Z = 0, then no movement
The gel used is divided into an upper "stacking" gel of low percentage (with large pore size) and low pH (6.8), where the protein bands get squeezed down as a thin layer migrating toward the anode and a resolving gel (pH 8.8) with smaller pores. Cl - is the only mobile anion present in both gels. When electrophoresis begins, glycine present in the electrophoresis buffer, enters the stacking gel, where the equilibrium favors zwitterionic form with zero net charge. The glycine front moves through the stacking gel slowly, lagging behind the strongly charged, Cl- ions. Since these two current carrying species separate, a region of low conductivity, with high voltage drop, is formed between them. This zone sweeps the proteins through the large pores of the stacking gel, and depositing it at the top of the resolving gel as a narrow band.
Stacking gel interactions:
Stacking occurs by the differential migration of ionic species, which carry the electric current through the gel. When an electrical current is applied to the gel, the negatively charged molecules start migrating to the positively charged electrode. Cl- ions, having the highest charge/mass ratio move faster, being depleted and concentrated at anode end. SDS coated proteins has a higher charge/mass ratio than glycine so it moves fast, but slower than Cl-. When protein encounters resolving gel it slows the migration because of increased frictional resistance, allowing the protein to stack in the gel.
Resolving Gel Interactions:
When glycine reaches resolving gel it becomes negatively charged and migrates much faster than protein due to higher charge/mass ratio. Now proteins are the main carrier of current and separate according to their molecular mass by the sieving effect of pores in gel.
Materials Required For PAGE
Acrylamide solutions (for resolving & stacking gels).
Isopropanol / distilled water .
Gel loading buffer.
Running buffer.
Staining, destaining solutions.
Protein samples .
Molecular weight markers.
The equipment and supplies necessary for conducting SDS-PAGE includes:
An electrophoresis chamber and power supply.
Glass plates(a short and a top plate).
Casting frame .
Casting stand.
Combs .
Note:
- Gloves should be worn, while performing SDS-PAGE.
- To ensure proper alignment, all the requirements should be clean.
- Special attention should be paid while using acrylamide(since it is a neurotoxin).
Reagents
- 30% Polyacrylamide solution(29g acrylamide+1g bisacrylamide in 50 mL of water, dissolve completely using a magnetic stirrer, make the volume upto 100mL). Keep the solution away from sunlight.
- 1.5 M Tris, pH 8.8
- 1 M Tris, pH 6.8
- 10% SDS(10 g SDS in 100mL distilled water).
- 10% ammonium persulfate (0.1 g in 1 ml water). It should be freshly prepared.
- 10x SDS running buffer( pH ~8.3) - Take 60.6 g Tris base, 288g Glycine and 20g SDS in separate beakers and dissolve them using distilled water. When properly dissolved ,mix three of them and make upto 2L.(working standard is 1X buffer).
Appendix 1:
Resolving gel (10%) Stacking gel (5%)
| dH20 |
4.0 ml |
| 30% acrylamide mix |
3.3 ml |
| 1.5M Tris pH8.8 |
2.5 ml |
| 10% SDS |
0.1 ml |
| 10% ammonium persulfate |
0.1 ml |
| TEMED |
0.004ml |
|
| dH20 |
5.65 ml |
30% acrylamide mix
|
1.65 ml |
1.0M Tris pH 6.8
|
2.5 ml |
10% SDS
|
0.1 ml |
10% ammonium persulfate
|
0.1 ml |
TEMED
|
0.004ml |
|
Gel loading buffer:
To make 10 mL of 4X stock:
2.0 ml 1M Tris-HCl pH 6.8.
0.8 g SDS.
4.0 ml 100% glycerol.
0.4 ml 14.7 M β-mercaptoethanol.
1.0 ml 0.5 M EDTA.
8 mg bromophenol Blue.
Staining solution:
Weigh 0.25g of Coomassie Brilliant Blue R250 in a beaker. Add 90 ml methanol:water (1:1 v/v) and 10ml of Glacial acetic acid ,mix properly using a magnetic stirrer. (when properly mixed, filter the solution through a Whatman No. 1 filter to remove any particulate matter and store in appropriate bottles)
Destaining solution:
Mix 90 ml methanol:water (1:1 v/v) and 10ml of Glacial acetic acid using a magnetic stirrer and store in appropriate bottles.
