polyacrylamide gel electrophoresis

Polyacrylamide Gel Electrophoresis(PAGE)

by Abhi Giri

Acrylamide

• Acrylamide CF- C3H5NO• White odourless crystalline solid, soluble in water,

ethanol,ether & chloroform• Prepared on industrial scale by the hydrolysis

of acrylonitrile by nitrile hydratase• carcinogenic as well as Neurotoxic compound.• used in the manufacture of dyes, Waste water

treatment and other monomers

Polyacrylamide• Also called Cross-linked Polyacrylamide

• Polyacrylamide is not toxic

• Polyacrylamide is a cross-linked polymer of Acrylamide • It is recommended to handle it with caution

• It is highly water-absorbent, forming a soft gel when hydrated

• Used in-- Flocculate or coagulate solids in a liquid- A subdermal filler for aesthetic facial surgery - Polyacrylamide gel electrophoresis- In soft contact lenses etc.

Polyacrylamide gel

• It is a white odorless gel, soluble in water

• After polymerization of acrylamide it get cross-linked structure

• TEMED stabilizes free radicals and improves polymerization

• Here, the toxic affect of acrylamide get vanish (95%)

• Amount of polyacrylamide salt dissolved (conc.) is directly proportion to cross –linked nature of gel

Gel Types

• Polysaccharide extracted from sea weed.

• Gel casted horizontally• Non-toxic.• Separate large molecules• Commonly used for DNA

separations.• Staining can be done before

or pouring the gel.

• Cross-linked polymer of acrylamide.

• Gel casted vertically.• Potent neuro-toxic.• Separate small molecules.• Used for DNA or protein

separations.• Staining can be done after

pouring the gel.

Agarose Polyacrylamide Gel

What is Gel Electrophoresis?

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

Electrophoresis• Electrophoresis is a separation technique that is

based on the movement of charged particles in an electric field.

• The term electrophoresis was coined from a Greek word “Phoresis” which means “Being Carried Away”.

• Hence literal meaning of the word electrophoresis means “to carry with electricity.”

• This electro kinetic phenomenon was observed for the first time in 1807 by Reuss.

PRINCIPLE• Any charged ion or molecule

migrates when placed in an electric field, the rate of migration depend upon its net charge, size, shape and the applied electric current.

• Can be represented by following eq.

ELECTROPHORESIS INSTRUMENTATION

Poly Acrylamide Gel Electrophoresis

• It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support matrix.

• Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.

• It is the most widely used technique of electrophoresis.

• A typical setup consists of a gel slab sandwiched between two glass plates, with the ends enclosed in upper and lower reservoirs of buffer

• Samples to be run are loaded in wells at the top of the gel, in conjunction with tracking dye. An electrical voltage is applied between the upper and lower reservoirs, causing the samples to migrate down through the gel.

Procedure of SDS-PAGE

Procedure

Set up Gel Load the Buffer

Load Sample

Assembling the glass plates:

•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.•Embed them into the casting frame and clamp them properly Make sure that the that the bottom ends of the glass plates are properly aligned.•Then place it on the casting stand.

Casting the gels2. Prepare 10%of resolving gel and 4.5% of stacking gel. •Prepare the separating gel solution by combining all reagents. Do not add Ammonium persulfate and TEMED.•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.)•Allow the gel to polymerize for 20-30 minutes .•Prepare stacking gel. Mix all reagents except APS and TEMED. Drain the isopropanol with strips of filter paper .•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.)•Place a comb in the stacking gel sandwich. Allow it to polymerize for 10 minutes.

Contents of Gel

Preparation of samples 3. 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 •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).•When the plates are secured, place them in the cassette and then lock it.•Place them in the gel running tank.

• Fill the inner chamber of the tank with buffer.(Now it is easy to remove the comb, since it is lubricated).

• Remove the comb CAREFULLY (without breaking the well).[Now the gel is ready to load the samples]

• Rinse the loading tip a few times with distilled water. (Make sure that all the water is poured out before loading the samples.)

• 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 .

• Attach the power supply by putting the lid (Make sure that the connection is in correct way i.e., black - black and red - red). Set the voltage up to 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.

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. •Filter through a Whatman No. 1 filter & store in bottles.

Destaining the Gel• 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).

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.

• Staining • Coomassie blue-sensitive to 0.1ug of protein• Silver- sensitive to 0.002ug of protein, based on ppt of silver ions

producing brown stain.

• greater sensitivity, radioactive samples can be used, allowing for exposure over time to produce images on photographic film, as seen in the sequencing gel on the right

• To calibrate the relative migrations of molecules of different size, a marker lane is often added, where samples of known size will migrate to reference positions

Types Of PAGE

• No denaturing agents• Proteins separated based on size charge and shape. • Used when want to keep protein active to study

conformation, self-association or aggregation, and the binding of other proteins

Native PAGE

SDS - PAGE• It is a modified version of PAGE whereby

Sodium-dodecyl-sulphate (SDS) is used.• SDS is an amphipathic surfactant. • It denatures proteins by binding to the protein

chain with its hydrocarbon ‘tail’, exposing normally buried regions and ‘coating’ the protein chain with surfactant molecules.

• The polar ‘head’ group of SDS adds an additional benefit to the use of this denaturant.

Structure of SDS

Differences

• Separation is based upon charge, size, and shape of macromolecules.

• Useful for separation and/or purification of mixture of proteins

• This was the original mode of electrophoresis.

• Separation is based upon the molecular weight of proteins.

• The most common method for determining MW of proteins

• Very useful for checking purity of protein samples

Native PAGE SDS PAGE

SDS-PAGE

WHY ? ? ?

• In their native form, proteins fold into a variety of shapes, some compact, some elongated.

• The rate of migration of native proteins through a sieving medium is therefore more a reflection of their relative compactness, and less an accurate measure of molecular weight.

• Denaturing the proteins nullifies structural

effects on mobility, allowing separation on a true charge/mass ratio basis.

• It also separates subunits in multimeric

proteins, allowing analysis of large, complex aggregates.

BEFORE SDS

AFTER SDS

Relative Mobility

Direction of movement is determined from Z: -

if Z < 0, then →+

if Z > 0, then → -

if Z = 0, then no movement

Rf is measured by:

where,

Z = charge on the molécule

E = Voltage applied

and ,

f = frictional resistance

Significance of SDS

• SDS is a anionic detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge

• For uniform distribution of charge per unit area(surface)(q/A)

• For getting the uniform direction of motion of molecules

• If a cell is incubated with SDS, the membranes will be dissolved and the proteins will be solubilized by the detergent

Advantages of PAGE

Applications• Used for estimation of molecular weight of proteins and

nucleic acids.• Determination of subunit structure of proteins.• Purification of isolated proteins.• Monitoring changes of protein content in body fluids. 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

Potential problems with Polyacrylamide gels– Under loaded (bands invisible)– Sloppy loading or to little concentration of protein – Bent bands– Tearing– frowning

1. Over loaded

2. Tearing

3. Frowning (run too hot)

4. Bent bands. Tearing

5. Sloppy loading or too low conc.

POLYACRYLAMIDE GEL ELECTROPHORESIS

References

• BIOCHEMISTRY by Donald Voet & Judith Voet, Wiley publications.

• BIOCHEMISTRY by Satyanarayana & Chakrapani.

• Biochemistry by Upadhyay , Upadhyay and Nath.

• http://amrita.vlab.co.in/?sub=3&brch=186&sim=319&cnt=2• http://www.protocolonline.org/prot/Molecular_Biology/

Electrophoresis/Polyacrylamide_Gel_Electrophoresis__PAGE_/index.html

• http://www.sciencedirect.com/science/article/pii/0003269760900361

• http://www.nature.com/nature physci/journal/v230/n12/abs/physci230092a0.html

polyacrylamide gel electrophoresis

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