C Biochemistry clinical practice CLS 432 Electrophoresis Dr. Samah Kotb Nasr El-deen C Biochemistry clinical practice CLS 432 Lecturer of Biochemistry 2015

Electrophoresis

Why electrophoresis? To separate DNA fragments from each other. To determine the sizes of DNA fragments. To determine the presence or amount of DNA. To analyze restriction digestion products. Determination of molecular weight of proteins.

What is electrophoresis? It describes migration of charged particles or molecules under the influence of electric field. It is standard method for separation, identification, analysis and purification of : DNA molecules. protein molecules.

PRINCIPLE separates molecules from each other on the basis of size charge shape basis of separation depends on how the sample and gel are prepared?

Gel electrophoresis What is a gel? Types of Gel: Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules. Types of Gel: Agarose gel. Polyacrylamide gel.

Agarose gel electrophoresis Agarose is extracted in the form of agar  from several species of red marine algae, or seaweed, found in California and eastern Asia,  dissolves in near-boiling water, and forms a gel when cools. Has gelling temperature 35-38˚C and melts at 90-95 ˚C. Used to separate macromolecules such as nucleic acids, large proteins and protein complexes.

Agarose gel electrophoresis(cont.) Composition: It is prepared by dissolving 0.5 % agarose in boiling water and allowing it to cool to 40°C. It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds.

Materials required for gel electrophoresis Electrophoresis chamber. Agarose gel. Gel casting tray. Buffer. Staining agent (dye). A comb . DNA ladder. Sample to be separate.

Electrophoresis Equipment

Gel Casting Trays available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.

Applied voltage  voltage,  rate of migration. The higher the voltage, the more quickly the gel runs. But if voltage is too high, gel melts. The best separation will apply voltage at no more than 5 V/cm of gel length.

Buffers During electrophoresis water undergoes hydrolysis : H2O  H + OH- . Buffers prevent the pH from changing by reacting with the H+ or OH- products. Most common buffer used is called TRIS [tris (hydroxymethyl) aminomethane].

Buffers (cont.) Another compound is added to make Tris an effective buffer — either boric or acetic acid. Another compound is added to bind metals EDTA. The buffer is either TBE or TAE. TBE is made with Tris/Boric Acid/EDTA. TAE is made with Tris/Acetic Acid/ EDTA.

Staining of DNA To make DNA fragments visible after electrophoresis, the DNA must be stained. The favorite—ethidium bromide. When bound to DNA it fluoresces under ultraviolet light (reddish –orange color).

Staining of DNA (cont.) Advantages of ethidium bromide 1. Convenient because it can be added directly to the gel. 2. Sensitive—detects 0.1ug of DNA. 3. Ethidium bromide is mutagenic so care must be taken while handling the dye. Other alternatives for ethidium bromide : Methylene blue. Syber safe. xylene cyanol. bromphenol blue.

A Comb A comb is placed in the liquid gel after it has been poured. Removing the comb from the hardened gel produces a series of wells used to load the DNA.

DNA ladder It is a solution of DNA molecules of different length. DNA ladder consists of known DNA sizes used to determine the size of an unknown DNA sample. The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a "ladder".

Method For Electrophoresis Prepare agarose gel Melt, cool and add Ethidium Bromide. Mix thoroughly. Pour into casting tray with comb and allow to solidify Add running buffer, load samples and marker Run gel at constant voltage until band separation occurs View DNA on UV light box and show results

Stain and look at with UV light Get your sample obtained from previous purifying technique (i.e. PCR) Load Buffer Set up gel Load Sample Run Gel Stain and look at with UV light

DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2 An agarose gel is used to slow the movement of DNA and separate by size. + - Power

Examples of serum proteins separation by gel electrophoresis

Serum protein electrophoresis Hydragel – agarose gel Serum proteins are separated into 6 groups: Albumin α1 - globulins α2 - globulins β1 - globulins β2 - globulins γ - globulins

Gels with 15 or 30 wells (serum samples) are used in laboratories of clinical biochemistry. Electrophoresis is also used for separation of isoenzymes,nucleic acids and immunoglobulins

Hypergamma Control Pictured 16-30 Normal Control Pictured 1-15

Serum proteins electrophoresis in diagnostics of diseases Normal pattern Reference ranges: Total protein 6.0 – 8.0 g/dL Albumin 3.5 – 5.0 g/dL α1-globulins 0.1 – 0.4 g/dL α2-globulins 0.4 – 1.3 g/dL β-globulins 0.6 – 1.3 g/dL γ-globulins 0.6 – 1.5 g/dL

Acute inflammatory response Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma normal or ↓ albumin ↑ α1 and α2 globulins α1 α2-globulins

Chronic inflammatory response α1 α2 γ-globulins Late response is correlated with chronic infection (autoimmune diseases, chronic liver disease, chronic infection, cancer) normal or ↓ albumin ↑α1 or α2 globulins ↑↑ γ globulins

Liver damage - Cirrhosis γ-globulins Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis ↓ albumin ↓ α1, α2 and β globulins ↑ Ig A

Nephrotic syndrome the kidney damage illustrates the long term loss of lower molecular weight proteins (↓ albumin and IgG – they are filtered in kidney) retention of higher molecular weight proteins (↑↑ α2-macroglobulin and ↑β-globulin) α2-globulin β-globulin fractions Figure is found at http://erl.pathology.iupui.edu/LABMED/INDEX.HTM