SDS PAGE

Objective:
To separate proteins on the basis of zxztheir 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).
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 zwitter ionic 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:

1. Gloves should be worn, while performing SDS-PAGE.

2. To ensure proper alignment, all the requirements should be clean.
3. Special attention should be paid while using acrylamide(since it is a neurotoxin).

Reagents

1. 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.
2. 1.5 M Tris, pH 8.8
3. 1 M Tris, pH 6.8
4. 10% SDS(10 g SDS in 100mL distilled water).
5. 10% ammonium persulfate (0.1 g in 1 ml water). It should be freshly prepared.
6. 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 5.65 ml
dH20 4.0 ml
30% acrylamide mix 1.65 ml
30% acrylamide mix 3.3 ml
1.5M Tris pH8.8 2.5 ml 1.0M Tris pH 6.8 2.5 ml
10% SDS 0.1 ml 10% SDS 0.1 ml
10% ammonium persulfate 0.1 ml 10% ammonium persulfate 0.1 ml
TEMED 0.004ml
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.

Procedure:
Assembling the glass plates:
1. Assemble the glass plate on a clean surface. Lay the longer glass plate(the one with spacer) down
first, then place the shorter glass plate on top of it.
2. Embed them into the casting frame and clamp them properly Make sure that the bottom ends of the
glass plates are properly aligned.
3. Then place it on the casting stand.

Casting the gels:

Prepare 10% resolving gel and 5% stacking gel.

NOTE: Please refer to appendix 1 for the recipe.

1. Prepare the separating gel solution by combining all reagents. Do not add Ammonium per sulfate and
TEMED.
2. Add APS and TEMED to the monomer solution(just before pouring ) and mix well by swirling gently.
Pour the solution till the mark. (It is ok if you introduce air bubbles, add a layer of isopropanol or
distilled water on top of the gel so as to level the poured gel.)
3. Allow the gel to polymerize for 20-30 minutes .
4. Prepare stacking gel. Mix all reagents except APS and TEMED. Drain the isopropanol with strips of
filter paper.
5. Add APS and TEMED to the monomer solution(just before pouring ) and mix well by swirling gently.
(Make sure you keep the comb ready by the side.)
6. Place a comb in the stacking gel sandwich. Allow it to polymerize for 10 minutes.

Preparation of samples:
Mix your protein in the ratio 4:1 with the sample buffer. Heat your sample by either:
 a) Boiling for 5-10 minutes. (Works for most proteins.)
b) 65°C for 10 minutes.
c) 37°C for 30 minutes.

Running the gel:

Note : Before running the gel make sure that the gel, gel apparatus and samples are ready.

1. To assemble, take out the gels from the casting frame and clamp them in the gel apparatus.( Make
sure that the short plate always faces inside and if you have got only one gel to run use the dummy
plate that is available to balance).
2. When the plates are secured, place them in the cassette and then lock it.
3. Place them in the gel running tank.
4. Fill the inner chamber of the tank with buffer. (Now it is easy to remove the comb, since it is
lubricated).
5. Remove the comb CAREFULLY (without breaking the well).
[Now the gel is ready to load the samples]
6. Rinse the loading tip a few times with distilled water. (Make sure that all the water is poured out
before loading the samples.)
7. Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse
the syringe with distilled water after loading for a few times.
8. Attach the power supply by putting the lid (Make sure that the connection is in correct way ie., black -
black and red - red). Set the voltage upto 180 V and run for 1 hour. (Don't allow the dye front to go
out of the gel).

Staining the gel:

After running, switch off the power supply and take out the gel plates, remove the gel. Place the gel in
the staining solution for 30 minutes. Destain the gel until the bands are properly seen. Determine the
approximate molecular weight of the visualised protein bands by comparing them with the molecular
weight ladders(markers).

Results and Discussion

It will be done in the class.

Applications:
a) To identify whether a particular protein is pure or not.
b) Separation of proteins, prior to Western Blot transfer.
c) Species identification.
d) Antigen preparation.
e) To measure genetic diversity.
VIDEO DEMONSTRATION

https://www.youtube.com/watch?v=EDi_n_0NiF4

https://www.youtube.com/watch?v=pnBZeL8nFEo