Gel ElectrophoresisBy: Tasnuva JhileekDr. Francine Norflus

Biotechnology

What is Gel Electrophoresis?

A procedure that separates molecules based on size and charge.

Uses an electric field

Molecules move through gel made of agar or polyacrylamide

Simple Procedure

Molecules are dispensed into a well in the gel material.

Gel is placed in an electrophoresis apparatus

Electric current is applied to the contents of the apparatus

In the gel: Larger molecules move more slowly Smaller molecules move faster

The different sized molecules form distinct bands on the gel.

Types of Gel

Agarose – DNA and protein

Polyacrylamide – Protein and DNA

Starch - protein

Agarose Gel Easy to cast, handle, store or dispose

Used to separate DNA fragments ranging from 50 base pair to several megabases (millions of bases).

Distance between DNA bands is determined by the percent agarose in the gel.

0.7% (large 5–10kb DNA fragments) and 2% (small 0.2–1kb fragments) agarose.

Low percentage gels - very weak and may break

High percentage gels - brittle and don’t set evenly

Polyacrylamide used for separating proteins ranging in size from 5 to

2,000 kDa - Uniform pore size

Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.

used to separate different proteins or isoforms of the same protein into separate bands and small DNA

gels are made in 6%, 8%, 10%, 12% or 15%.

percentage chosen depends on the size of the protein smaller weight = higher gel

Starch

Partially hydrolysed potato starch – protein electrophoresis

Non-denatured proteins are separated according to charge and size.

They are visualised using Napthal Black or Amido Black staining.

Typical starch gel concentrations are 5% to 10%

Nucleic Acid Electrophoresis analytical technique used to separate DNA or RNA fragments by

size.

an electric field induces the nucleic acids to migrate toward the anode

smaller fragments end up nearer to the anode

DNA is frequently cut into smaller fragments using a DNA restriction endonuclease (or restriction enzyme) or even PCR

Fragment size determination is typically done by comparison to commercially available DNA markers containing linear DNA fragments of known length

agarose (for relatively long DNA molecules) and polyacrylamide (for high resolution of short DNA molecules

Protein Electrophoresis proteins, unlike nucleic acids, can have varying charges and

complex shapes

proteins are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP)

the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

Protein Electrophoresis cont.

Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

Gel Condition

Denature - the native structure of macromolecules that are run within the gel is not maintained. denatures the native structure of a

protein

Native - separation method typically used in proteomics and metallomics. does not use a charged denaturing agent.

Buffers

nucleic acids - Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE).

TAE - lowest buffering capacity but best resolution for larger DNA. This means a lower voltage and more time, but a better product.

Xylene cyanol and Bromophenol blue are common dyes found in loading buffers

Visualization

DNA may be visualized using ethidium bromide

SYBR Green I – more expensive, quicker, safer

Ethidium Bromide

light sensitive and is stored in a brown bottle covered in tin foil (aluminum foil).

fluoresces under UV light when intercalated into the major groove of DNA (or RNA)

any band containing more than ~20 ng DNA becomes distinctly visible

protein may be visualised using silver stain or Coomassie Brilliant Blue dye.

Mutagen

ALWAYS wear gloves

Areas of Application

used in forensics, molecular biology, genetics, microbiology and biochemistry.

results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device

the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel.

the measurement and analysis are mostly done with specialized software.

Lab Procedure Make Ethidium Bromide

Make buffer

Add agarose to buffer and microwave

Pour gel after setting comb

Pour remaining buffer + EtBr in apparatus

Load gel (Black to Red 60 – 100 V)

Run gel

Kodak Moment

Discard waste product safely

Making EtBr

1 g of Ethidium Bromide in 100 ml of dH20 in DARK colored bottle of transparent glass bottle wrapped in aluminum foil.

Dissolve completely (use Stir bar)

Use caution

Wear gloves at all times

Discard all EtBr related waste to biohazard

Making Buffer

Calculation: Total of 300 ml of solution

15 ml of TBE + 285 ml dH2O

Making Gel

Calculations: 0.8% agarose gel in 100 ml of solution

Mix 0.8 g of agarose gel in 100 ml of buffer solution.

Microwave until agarose dissolves completely in buffer.

Let it cool (not solidify).

Pour!

Preparing the DNA sample

10 microliter DNA + 2 microliter tracking dye in microcentrifuge tube.

Ladder: 1 microliter concentrated ladder per 9 microliter sterile water

Load all contents in separate wells in gel.

Setting up the apparatus

5 microliter Ethidium Bromide + 100 microliter TBE solution in electrophoresis apparatus

Plug apparatus into the electric outlet

Put gel in the apparatus (wells being in the black side)

DNA will travel from Black to Red

Set V to 60 or 100

Run DNA for about 30-40 minutes

What is the safe way to discard the waste?

Put all EtBr related waste in the biohazard bin.

Discard buffer from the apparatus into biohazard bottle.

Wash gel in sink before throwing in the trash